Combination treatment of cd38-expressing tumors

ABSTRACT

The invention relates to novel method for the treatment of cancer using a combination therapy comprising an antibody that binds CD38, a corticosteroid and a non-corticosteroid chemotherapeutic agent.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/566,279, filed Dec. 10, 2014, which is a continuation of U.S. patentapplication Ser. No. 12/442,808, filed May 11, 2009 (now U.S. Pat. No.9,040,050), which is a 35 U.S.C. 371 national stage filing ofInternational Application No. PCT/DK2007/000418, filed Sep. 26, 2007,which claims priority to, and the benefit of, Denmark Patent ApplicationNo. PA 2006/01232, filed Sep. 26, 2006, and U.S. Provisional ApplicationNo. 60/847,329, filed Sep. 26, 2006. The aforementioned applications arehereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 12, 2019, isnamed GMI_100USCNDV_Sequence_Listing.txt and is 31,831 bytes in size.

FIELD OF THE INVENTION

The present invention relates to the treatment of cancer using acombination therapy comprising an antibody that binds CD38, acorticosteroid and a non-corticosteroid chemotherapeutic agent.

BACKGROUND

Multiple myeloma is a B cell malignancy characterized by the latentaccumulation in bone marrow of secretory plasma cells with a lowproliferative index and an extended life span. The disease ultimatelyattacks bones and bone marrow, resulting in multiple tumors and lesionsthroughout the skeletal system.

Approximately 1% of all cancers, and slightly more than 10% of allhematologic malignancies, can be attributed to multiple myeloma (MM).Incidence of MM increases in the aging population, with the median ageat time of diagnosis being about 61 years.

Currently available therapies for multiple myeloma include chemotherapy,stem cell transplantation, Thalomid® (thalidomide), Velcade®(bortezomib), Aredia® (pamidronate), and Zometa® (zoledronic acid).Current treatment protocols, which include a combination ofchemotherapeutic agents such as vincristine, BCNU, melphalan,cyclophosphamide, adriamycin, and prednisone or dexamethasone, yield acomplete remission rate of only about 5%, and median survival isapproximately 36-48 months from the time of diagnosis. Recent advancesusing high dose chemotherapy followed by autologous bone marrow orperipheral blood mononuclear cell transplantation have increased thecomplete remission rate and remission duration. Yet overall survival hasonly been slightly prolonged, and no evidence for a cure has beenobtained. Ultimately, all MM patients relapse, even under maintenancetherapy with interferon-alpha (IFN-α) alone or in combination withsteroids.

If a patient is candidate or possible candidate for autologoustransplant, induction therapy often involve non-alkylating chemotherapy,in that alkylating agents interfere with harvesting (stem cellcollection). The preferred regimen is VAD, which allows for subsequentharvest (Wu K L, Clin Lymphoma Myeloma 2005; 6:96). Another treatmentmodality, tested in induction setting before transplant, includesThalidomide combined with dexamethasone (Cavo M Blood 2005; 106:35).

Efficacy of the available chemotherapeutic treatment regimens for MM islimited by the low cell proliferation rate and development of multi-drugresistance. For more than 90% of MM patients, the disease becomeschemoresistant. As a result, alternative treatment regimens aimed atadoptive immunotherapy targeting surface antigens on plasma cells arebeing sought.

CD38 is an example of an antigen expressed on such malignant plasmacells, and is expressed in a variety of malignant hematologicaldiseases, including but not restricted to, multiple myeloma, B-cellchronic lymphocytic leukemia, B-cell acute lymphocytic leukemia,Waldenström macroglobulinemia, primary systemic amyloidosis, mantle-celllymphoma, pro-lymphocytic/myelocytic leukemia, acute myeloid leukemia,chronic myeloid leukemia, follicular lymphoma, NK-cell leukemia andplasma-cell leukemia. Expression of CD38 has been described onepithelial/endothelial cells of different origin, including glandularepithelium in prostate, islet cells in pancreas, ductal epithelium inglands, including parotid gland, bronchial epithelial cells, cells intestis and ovary and tumor epithelium in colorectal adenocarcinoma.Diseases where CD38 expression could be involved, include but are notrestricted to broncho-epithelial carcinomas of the lung, breast cancer(evolving from malignant proliferation of epithelial lining in ducts andlobules of the breast), pancreatic tumors, evolving from the b-cells(insulinomas), tumors evolving from epithelium in the gut (e.g.adenocarcinoma and squamous cell carcinoma) In CNS, neuroblastomasexpress CD38. Other such diseases include carcinoma in the prostategland, seminomas in testis and ovarian cancers.

Normally, CD38 is expressed by hemopoietic cells, and in solid tissues.With regard to hemopoietic cells, the majority of medullary thymocytesare CD38+, resting and circulating T- and B-cells are CD38-, andactivated cells are CD38+. CD38 is also expressed on approximately 80%of resting NK cells and monocytes, and on lymph node germinal centerlymphoblasts, plasma B cells and some intrafollicular cells. CD38 canalso be expressed by dendritic cells. A significant proportion of normalbone marrow cells, particular precursor cells, express CD38. In additionto lymphoid precursor cells, CD38 is also expressed on erythrocytes andon platelets.

With regard to solid tissues, CD38 is expressed in the gut byintra-epithelial cells and lamina propria lymphocytes, by Purkinje cellsand neurofibrillary tangles in the brain, by epithelial cells in theprostate, β-cells in the pancreas, osteoclasts in the bone, retinalcells in the eye, and sarcolemma of smooth and striated muscle.

Functions ascribed to CD38 include both receptor mediation in adhesionand signaling events and (ecto-) enzymatic activity. As an ectoenzyme,CD38 uses NAD⁺ as substrate for the formation of cyclic ADP-ribose(cADPR) and ADPR, but also of nicotinamide and nicotinic acid-adeninedinucleotide phosphate (NAADP). cADPR and NAADP have been shown to actas second messengers for Ca²⁺ mobilization. By converting NAD+ to cADPR,CD38 regulates the extracellular NAD+ concentration and hence cellsurvival by modulation of NAD-induced cell death (NCID). In addition tosignaling via Ca²⁺, CD38 signaling occurs via cross-talk withantigen-receptor complexes on T and B cells or other types of receptorcomplexes, e.g. MHC molecules, and is in this way involved in severalcellular responses, but also in switching and secretion of IgG1.

Anti-CD38 antibodies are described in the literature, for instance inLande R, et al., Cell Immunol. 220(1), 30-8 (2002), Ausiello C M, etal., Tissue Antigens. 56(6), 539-47 (2000), and Cotner T, et al., Int JImmunopharmacol. 3(3), 255-68 (1981) and in WO2005/103083 (Morphosys).CD38 has a number of functions, which may or may not be activated by amolecule binding to CD38. For instance the mouse anti-CD38 antibody IB4has agonistic properties in relation to CD38. IB4 is shown to induce Tcell activation as indicated by Ca²⁺ mobilization in Jurkat cells(Zubiaur M, et al., J Immunol. 159(1), 193-205 (1997), to inducesignificant proliferation of peripheral blood mononuclear cells (PBMCs),to induce release of significant IL-6 levels and to induce release ofdetectable IFN-γ levels (Lande, Zubiaur Morra, Ansiello supra).

It is clear that in spite of the recent progress in the discovery anddevelopment of anti-cancer agents, many forms of cancer involvingCD38-expressing tumors still have a poor prognosis. Thus, there is aneed for improved methods for treating such forms of cancer.

SUMMARY OF THE INVENTION

It is an object of the invention to provide improved methods for thetreatment of CD38-expressing tumors that result in increased efficacyand/or prolonged survival.Thus, in a first main aspect, the invention relates to a method forinhibiting growth and/or proliferation of tumor cells expressing CD38 inan individual in need thereof, which method comprises administration tothe said individual of

i) a non-agonistic antibody which binds to CD38,

ii) at least one corticosteroid, and

iii) at least one non-corticosteroid chemotherapeutic agent.

The three types of medicaments may be administered simultaneously orsequentially in any order. Furthermore, they may be administeredseparately or in one or two pharmaceutical compositions.The triple therapy may, in some embodiments, allow administration oflower amounts of a medicament than when used as mono- or in duplextherapy. Such lower amounts may generate fewer side-effects, allowingmore effective treatment of patients that cannot be treated with highdoses, such as elderly or hypersensitive patients.In one embodiment, the non-agonistic antibody which binds to CD38 usedin the invention is antibody -005, -003 or -024, described herein. Theseantibodies have previously been described in patent applicationPCT/DK2006/000166 (WO 2006099875) (Genmab).In some embodiments, said at least one non-corticosteroidchemotherapeutic agent comprises

-   -   an alkylating agent, such as melphalan,

and/or

-   -   a glutamic acid derivative, such as thalidomide or lenalidomide

and/or

-   -   a proteasome inhibitor, such as bortezomib.        In a similar aspect, the invention relates to a method of        treating cancer involving cells expressing CD38 in an        individual, wherein said method comprises the features of the        method described above.        In a further aspect, the invention relates to a method for        treating cancer involving tumor cells expressing CD38 in an        individual in need thereof, which method comprises        administration to the said individual of:

i) a non-agonistic antibody which binds to CD38,

ii) optionally at least one corticosteroid, and

iii) optionally at least one non-corticosteroid chemotherapeutic agent,followed by autologous peripheral stem cell or bone marrowtransplantation.

Thus, in this method, the anti-CD38 antibody is used in inductiontherapy preceding autologous peripheral stem cell or bone marrowtransplantation. Without being bound by any specific theory, it isbelieved that anti-CD38 antibodies are particularly suitable for suchinduction therapy, because they do not have many undesired side-effects,thus keeping the patient in good condition before the transplant.

In an even further aspect, the invention relates to a therapeuticcombination for inhibiting growth and/or proliferation of tumor cellsexpressing CD38, comprising

i) a non-agonistic antibody which binds to CD38,

ii) at least one corticosteroid, and

iii) at least one non-corticosteroid chemotherapeutic agent,

wherein the combination is suitable for separate, sequential and/orsimultaneous administration.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the binding of -003, -005 and the isotype control antibodyHuMab-KLH to CD38-transfected CHO (CHO-CD38) cells as measured by flowcytometry. The experimental setup is described in Example 4.

FIG. 1B shows the binding of -024 and HuMab-KLH to CD38-transfected CHO(CHO-CD38) cells as measured by flow cytometry. The experimental setupis described in Example 4.

FIG. 2A shows the binding of -003, -005 and HuMab-KLH to Daudi cells asmeasured by flow cytometry. The experimental setup is described inExample 4.

FIG. 2B shows the binding of -024 and HuMab-KLH to Daudi cells asmeasured by flow cytometry. The experimental setup is described inExample 4.

FIG. 3 shows the binding of -003, -005, -024 and HuMab-KLH to multiplemyeloma cells. The experimental setup is described in Example 4.

FIG. 4A shows the ability of -003 and -005 to induce lysis of Daudicells by ADCC as compared to rituximab and HuMab-KLH. The experimentalsetup is described in Example 5.

FIG. 4B shows the ability of -024 to induce lysis of Daudi cells by ADCCas compared to HuMab-KLH. The experimental setup is described in Example5.

FIG. 5A shows the ability of -003, -005 and -024 to induce lysis offresh multiple myeloma tumor cells by ADCC as compared to HuMab-KLH. Theexperimental setup is described in Example 5.

FIG. 5B shows the ability of -003, -005 and -024 to induce lysis offresh plasma cell leukemia tumor cells by ADCC as compared to HuMab-KLH.The experimental setup is described in Example 5.

FIG. 6 shows the ability of -003 and -005 to induce lysis of JK6L (amultiple myeloma cell line) by ADCC as compared to HuMab-KLH. Theexperimental setup is described in Example 5.

FIG. 7 shows the ability of -003 and -005 to induce lysis of AMO-1 (amultiple myeloma cell line) by ADCC as compared to HuMab-KLH. Theexperimental setup is described in Example 5.

FIG. 8 shows the CDC-mediated lysis of Daudi-luc cells induced by -003and -005 compared to HuMab-KLH. The experimental setup is described inExample 6.

FIG. 9A shows the CDC-mediated lysis of CHO-CD38 cells induced by -003and -005 compared to HuMab-KLH. The experimental setup is described inExample 6.

FIG. 9B shows the CDC-mediated lysis of CHO-CD38 cells induced by -024compared with HuMab-KLH. The experimental setup is described in Example6.

FIG. 10A shows the CDC-mediated lysis of 3% refractory tumor cells inthe presence of -003, -005 and HuMab-KLH. The experimental setup isdescribed in Example 6.

FIG. 10B shows the CDC-mediated lysis of 9% refractory tumor cells inthe presence of -003, -005 and HuMab-KLH. The experimental setup isdescribed in Example 6.

FIG. 10C shows the CDC-mediated lysis of 30-40% tumor cells in thepresence of -003, -005 and HuMab-KLH. The experimental setup isdescribed in Example 6.

FIG. 10D shows the CDC-mediated lysis of 70% tumor cells in the presenceof -003, -005 and HuMab-KLH. The experimental setup is described inExample 6.

FIG. 10E shows the CDC-mediated lysis of multiple myeloma cells in thepresence of -024 and HuMab-KLH. The experimental setup is described inExample 6.

FIG. 11 shows that -003 and -005 do not cross-block binding to CD38. Theexperimental setup is described in Example 7.

FIG. 12A shows the immunohistological staining of macrophages,lymphocytes and plasma B cells with -003. The experimental setup isdescribed in Example 10.

FIG. 12B shows the immunohistological staining of bronchial epitheliumwith -003. The experimental setup is described in Example 10.

FIG. 12C shows the immunohistological staining of myocytes with -003.The experimental setup is described in Example 10.

FIG. 12D shows the immunohistological staining of cynomolgus lymphoidtissue with -003. The experimental setup is described in Example 10.

FIG. 13A shows the immunohistological staining of macrophages,lymphocytes and plasma B cells with -005. The experimental setup isdescribed in Example 10.

FIG. 13B shows the immunohistological staining of bronchial epitheliumwith -005. The experimental setup is described in Example 10.

FIG. 13C shows the immunohistological staining of myocytes with -005.The experimental setup is described in Example 10.

FIG. 13D shows the immunohistological staining of cynomolgus lymphoidtissue with -005. The experimental setup is described in Example 10.

FIG. 14A shows immunohistological staining of liver endothelium withCD31. The experimental setup is described in Example 10.

FIG. 14B shows immunohistological staining of liver endothelium withvWF. The experimental setup is described in Example 10.

FIG. 14C shows immunohistological staining of liver endothelium withanti-KLH. The experimental setup is described in Example 10.

FIG. 14D shows immunohistological staining of liver endothelium with-003. The experimental setup is described in Example 10.

FIG. 14E shows immunohistological staining of liver endothelium with-005. The experimental setup is described in Example 10.

FIG. 15A shows the cross-reactivity of -003 and -005 compared toHuMab-KLH on cynomolgus lymphocytes as measured by flow cytometry. Theexperimental setup is described in Example 11.

FIG. 15B shows the cross-reactivity of -003 and -005 compared toHuMab-KLH on cynomolgus monocytes as measured by flow cytometry. Theexperimental setup is described in Example 11.

FIG. 15C shows the cross-reactivity of -003 and -005 compared toHuMab-KLH on rhesus monkey PBMCs as measured by flow cytometry. Theexperimental setup is described in Example 11.

FIG. 16A shows the internalization of -003 as measured byEtBr-quenching. The experimental setup is described in Example 12.

FIG. 16B shows the internalization of -005 as measured byEtBr-quenching. The experimental setup is described in Example 12.

FIG. 17A shows the inhibition caused by -003 and -005 compared to ananti-CD20 monoclonal antibody (rituximab) and HuMab-KLH of the growth oftumor cells in a preventive setting as measured by in vivo SCIDluciferase imaging. The experimental setup is described in Example 13.

FIG. 17B shows the inhibition caused by -003 and -005 compared to ananti-CD20 monoclonal antibody (rituximab) and HuMab-KLH of the growth oftumor cells in therapeutic setting I as measured by in vivo SCIDluciferase imaging. The experimental setup is described in Example 13.

FIG. 17C shows the inhibition caused by -003 and -005 compared to ananti-CD20 monoclonal antibody (rituximab) and HuMab-KLH of the growth oftumor cells in therapeutic setting II as measured by in vivo SCIDluciferase imaging. The experimental setup is described in Example 13.

FIG. 17D shows the inhibition of tumor cell growth by -003 and -024compared to HuMab-KLH in therapeutic setting III as measured by in vivoSCID luciferase imaging. The experimental set up is described in Example13.

FIG. 18 shows the induction of apoptosis by -003 and -005 compared to ananti-CD20 monoclonal antibody (rituximab) and HuMab-KLH without or withcross-linking. The experimental setup is described in Example 14.

FIG. 19 shows the histological score for CD38-positive cells inimplanted RA-SCID mouse xenografts on day 14, after treatment withanti-KLH (HuMab-KLH) or -005. Methods are described in Example 15.

FIG. 20 shows the histological score for CD138-positive cells inimplanted RA-SCID mouse xenografts on day 14, after treatment withanti-KLH or -005. Methods are described in Example 15.

FIGS. 21A-21C show CD38 staining of B cells in xenografts beforeimplantation (FIG. 21A), or after treatment with anti-KLH (FIG. 21B), or-005 (FIG. 21C). Methods are described in Example 15.

FIGS. 22A-22C show CD138 staining of B cells in xenografts beforeimplantation (FIG. 22A), or after treatment with anti-KLH (FIG. 22B), or-005 (FIG. 22C). Methods are described in Example 15.

FIGS. 23A-23C show the binding of -003 and -005 to wild type and mutanthuman CD38 as measured by ELISA. FIG. 23A: Binding of -003 and -005 toT237A mutant human CD38. FIG. 23B: Binding of -003 and -005 to Q272Rmutant human CD38. FIG. 23C: Binding of -003 and -005 to S274F mutanthuman CD38. Methods are described in Example 17.

FIGS. 24A-24C show the effect of -003 and -005 compared to HuMab-KLH onproliferation (FIG. 24A), IL-6 production (FIG. 24B) and IFN-γproduction (FIG. 24C) of human PBMCs. Methods are described in Examples18, 19 and 20, respectively.

FIGS. 25A-25D show the enzymatic production of cGDPribose in thepresence of various concentrations of -003 (FIG. 25B), -005 (FIG. 25C),-024 (FIG. 25D) or anti-KLH (FIG. 25A). Methods are described in Example23.

FIGS. 26A-26C show the comparison between -003, -005 and Morphosysantibody TH-3079 in CDC of CHO-CD38 cells (FIG. 26A), CDC of Daudi cells(FIG. 26B), and ADCC of Daudi cells (FIG. 26C). Methods are described inExample 24.

FIG. 27 shows the binding of -005 and the isotype control antibodyHuMab-KLH to EBV transformed chimpanzee B cells as measured by flowcytometry. The experimental setup is described in Example 26.

FIG. 28 shows the capacity of -005 alone and in combination with othercompounds (Dexamethasone (Dex) and Bortezomib (Bor)) to induce celldeath of the multiple myeloma cell line UM6 in vitro.

FIG. 29 shows a sequence listing of the sequences of the invention.

-   -   SEQ ID No:1 is the nucleotide sequence of the V_(L) region of        the antibody -003.    -   SEQ ID No:2 is the amino acid sequence of the V_(L) region of        the antibody -003.    -   SEQ ID No:3 is the amino acid sequence of the V_(L) CDR1 of the        antibody -003 comprising aa 24-34 of SEQ ID No:2.    -   SEQ ID No:4 is the amino acid sequence of the V_(L) CDR2 of the        antibody -003 comprising aa 50-56 of SEQ ID No:2.    -   SEQ ID No:5 is the amino acid sequence of the V_(L) CDR3 of the        antibody -003 comprising aa 89-97 of SEQ ID No:2.    -   SEQ ID No:6 is the nucleotide sequence of the V_(H) region of        the antibody -003.    -   SEQ ID No:7 is the amino acid sequence of the V_(H) region of        the antibody -003.    -   SEQ ID No:8 is the amino acid sequence of the V_(H) CDR1 of the        antibody -003 comprising aa 31-35 of SEQ ID No:7.    -   SEQ ID No:9 is the amino acid sequence of the V_(H) CDR2 of the        antibody -003 comprising aa 50-66 of SEQ ID No:7.    -   SEQ ID No:10 is the amino acid sequence of the V_(H) CDR3 of the        antibody -003 comprising aa 99-109 of SEQ ID No:7.    -   SEQ ID No:11 is the nucleotide sequence of the V_(L) region of        the antibody -005.    -   SEQ ID No:12 is the amino acid sequence of the V_(L) region of        the antibody -005.    -   SEQ ID No:13 is the amino acid sequence of the V_(L) CDR1 of the        antibody -005 comprising aa 24-34 of SEQ ID No:12.    -   SEQ ID No:14 is the amino acid sequence of the V_(L) CDR2 of the        antibody -005 comprising aa 50-56 of SEQ ID No:12.    -   SEQ ID No:15 is the amino acid sequence of the V_(L) CDR3 of the        antibody -005 comprising aa 89-97 of SEQ ID No:12.    -   SEQ ID No:16 is the nucleotide sequence of the V_(H) region of        the antibody -005.    -   SEQ ID No:17 is the amino acid sequence of the V_(H) region of        the antibody -005.    -   SEQ ID No:18 is the amino acid sequence of the V_(H) CDR1 of the        antibody -005 comprising aa 31-35 of SEQ ID No:17.    -   SEQ ID No:19 is the amino acid sequence of the V_(H) CDR2 of the        antibody -005 comprising aa 50-66 of SEQ ID No:17.    -   SEQ ID No:20 is the amino acid sequence of the V_(H) CDR3 of the        antibody -005 comprising aa 99-111 of SEQ ID No:17.    -   SEQ ID No:21 is the nucleotide sequence of the V_(L) region of        the antibody -024.    -   SEQ ID No:22 is the amino acid sequence of the V_(L) region of        the antibody -024.    -   SEQ ID No:23 is the amino acid sequence of the V_(L) CDR1 of the        antibody -024 comprising aa 24-34 of SEQ ID No:22.    -   SEQ ID No:24 is the amino acid sequence of the V_(L) CDR2 of the        antibody -024 comprising aa 50-56 of SEQ ID No:22.    -   SEQ ID No:25 is the amino acid sequence of the V_(L) CDR3 of the        antibody -024 comprising aa 89-97 of SEQ ID No:22.    -   SEQ ID No:26 is the nucleotide sequence of the V_(H) region of        the antibody -024.    -   SEQ ID No:27 is the amino acid sequence of the V_(H) region of        the antibody -024.    -   SEQ ID No:28 is the amino acid sequence of the V_(H) CDR1 of the        antibody -024 comprising aa 31-35 of SEQ ID No:27.    -   SEQ ID No:29 is the amino acid sequence of the V_(H) CDR2 of the        antibody -024 comprising aa 50-66 of SEQ ID No:27.    -   SEQ ID No:30 is the amino acid sequence of the V_(H) CDR3 of the        antibody -024 comprising aa 99-111 of SEQ ID No:27.    -   SEQ ID No:31 is the sequence of human CD38.    -   SEQ ID No:32 is the sequence of a mutant human CD38, wherein the        threonine residue in position 237 has been substituted with an        alanine residue.    -   SEQ ID No:33 is the sequence of a mutant human CD38, wherein the        glutamine residue in position 272 has been substituted with an        arginine residue.    -   SEQ ID No:34 is the sequence of a mutant human CD38, wherein the        serine residue in position 274 has been substituted with a        phenylalanine residue.

DETAILED DESCRIPTION OF THE INVENTION Definitions

A “non-agonistic antibody which binds to CD38” or “anti-CD38 antibody”when used herein refer to an antibody which upon binding to CD38 doesnot induce significant proliferation of peripheral blood mononuclearcells when compared to the proliferation induced by an isotype controlantibody or medium alone (as assayed e.g. as described herein below inExample 18). In one embodiment, an anti-CD38 antibody used in theinvention is not only a non-agonist, but even an antagonist of CD38.

The terms “CD38” and “CD38 antigen” are used interchangeably herein, andinclude any variants, isoforms and species homologs of human CD38, whichare naturally expressed by cells or are expressed on cells transfectedwith the CD38 gene. Synonyms of CD38, as recognized in the art, includeADP ribosyl cyclase 1, cADPr hydrolase 1, Cd38-rs1, Cyclic ADP-ribosehydrolase 1, I-19, NIM-R5 antigen.

The term “immunoglobulin” refers to a class of structurally relatedglycoproteins consisting of two pairs of polypeptide chains, one pair oflight (L) low molecular weight chains and one pair of heavy (H) chains,all four inter-connected by disulfide bonds. The structure ofimmunoglobulins has been well characterized. See for instanceFundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)). Briefly, each heavy chain typically is comprised of a heavychain variable region (abbreviated herein as V_(H)) and a heavy chainconstant region. The heavy chain constant region typically is comprisedof three domains, C_(H)1, O_(H)2, and C_(H)3. Each light chain typicallyis comprised of a light chain variable region (abbreviated herein asV_(L)) and a light chain constant region. The light chain constantregion typically is comprised of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability (orhypervariable regions which can be hypervariable in sequence and/or formof structurally defined loops), also termed complementarity determiningregions (CDRs), interspersed with regions that are more conserved,termed framework regions (FRs).

Each V_(H) and V_(L) is typically composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol.Biol. 196, 901-917 (1987)). Typically, the numbering of amino acidresidues in this region is performed by the method described in Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)(phrases such as variable domain residue numbering as in Kabat oraccording to Kabat herein refer to this numbering system for heavy chainvariable domains or light chain variable domains). Using this numberingsystem, the actual linear amino acid sequence of a peptide may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of V_(H) CDR2 and insertedresidues (for instance residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The term “antibody” (Ab) in the context of the present invention refersto an immunoglobulin molecule, a fragment of an immunoglobulin molecule,or a derivative of either thereof, which has the ability to specificallybind to an antigen under typical physiological conditions forsignificant periods of time such as at least about 30 minutes, at leastabout 45 minutes, at least about one hour, at least about two hours, atleast about four hours, at least about 8 hours, at least about 12 hours,about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 ormore days, etc., or any other relevant functionally-defined period (suchas a time sufficient to induce, promote, enhance, and/or modulate aphysiological response associated with antibody binding to the antigen).

The variable regions of the heavy and light chains of the immunoglobulinmolecule contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies (Abs) may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (such as effector cells) and the first component (Clq)of the classical complement system.

An anti-CD38 antibody may be a bispecific antibody, diabody, or similarmolecule (see for instance PNAS USA 90(14), 6444-8 (1993) for adescription of diabodies). Indeed, bispecific antibodies, diabodies, andthe like, provided by the present invention may bind any suitable targetin addition to a portion of CD38.

As indicated above, the term antibody herein, unless otherwise stated orclearly contradicted by context, includes fragments of an antibody thatretain the ability to specifically bind to an antigen. It has been shownthat the antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antibody” include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H)1domains; (ii) F(ab)₂ and F(ab′)₂ fragments, bivalent fragmentscomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting essentially of the V_(H) andC_(H)1 domains; (iv) a Fv fragment consisting essentially of the V_(L)and V_(H) domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., Nature 341, 544-546 (1989)), which consists essentially ofa V_(H) domain; (vi) an isolated complementarity determining region(CDR), and (vii) a combination of two or more isolated CDRs which mayoptionally be joined by a synthetic linker. Furthermore, although thetwo domains of the Fv fragment, V_(L) and V_(H), are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the V_(L) and V_(H) regions pair to form monovalent molecules(known as single chain antibodies or single chain Fv (scFv), see forinstance Bird et al., Science 242, 423-426 (1988) and Huston et al.,PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies areencompassed within the term antibody unless otherwise noted or clearlyindicated by context. Other forms of single chain antibodies, such asdiabodies are included within the term antibody. Although such fragmentsare generally included within the meaning of antibody, they collectivelyand each independently are unique features of the present invention,exhibiting different biological properties and utility. These and otheruseful antibody fragments in the context of the present invention arediscussed further herein.

It also should be understood that the term antibody also generallyincludes polyclonal antibodies, monoclonal antibodies (mAbs),antibody-like polypeptides, such as chimeric antibodies and humanizedantibodies, anti-idiotypic (anti-Id) antibodies to antibodies, andantibody fragments retaining the ability to specifically bind to theantigen (antigen-binding fragments) provided by any known technique,such as enzymatic cleavage, peptide synthesis, and recombinanttechniques. An antibody as generated can possess any isotype.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents. The epitope may comprise amino acid residues directly involvedin the binding (also called immunodominant component of the epitope) andother amino acid residues, which are not directly involved in thebinding, such as amino acid residues which are effectively blocked bythe specifically antigen binding peptide (in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide).

The term “bispecific molecule” is intended to include any agent, such asa protein, peptide, or protein or peptide complex, which has twodifferent binding specificities. For example, the molecule may bind to,or interact with, (a) a cell surface antigen and (b) an Fc receptor onthe surface of an effector cell. The term “multispecific molecule” isintended to include any agent, for instance a protein, peptide, orprotein or peptide complex, which has more than two different bindingspecificities. For example, the molecule may bind to, or interact with,(a) a cell surface antigen, (b) an Fc receptor on the surface of aneffector cell, and (c) at least one other component. Accordingly, thepresent invention includes, but is not limited to, bispecific,trispecific, tetraspecific, and other multispecific molecules which aredirected to CD38, and to other cell surface antigens or targets, such asFc receptors on effector cells.

The term “bispecific antibodies” is intended to include any anti-CD38antibody, which is a bispecific molecule. The term “bispecificantibodies” also includes diabodies. Diabodies are bivalent, bispecificantibodies in which the V_(H) and V_(L) domains are expressed on asingle polypeptide chain, but using a linker that is too short to allowfor pairing between the two domains on the same chain, thereby forcingthe domains to pair with complementary domains of another chain andcreating two antigen binding sites (see for instance Holliger, P. etal., PNAS USA 90, 6444-6448 (1993), Poljak, R. J. et al., Structure 2,1121-1123 (1994)).

As used herein, the term “effector cell” refers to an immune cell whichis involved in the effector phase of an immune response, as opposed tothe cognitive and activation phases of an immune response. Exemplaryimmune cells include a cell of a myeloid or lymphoid origin, forinstance lymphocytes (such as B cells and T cells including cytolytic Tcells (CTLs)), killer cells, natural killer cells, macrophages,monocytes, eosinophils, neutronphils, polymorphonuclear cells,granulocytes, mast cells, and basophils. Some effector cells expressspecific Fc receptors and carry out specific immune functions. In someembodiments, an effector cell is capable of inducing antibody-dependentcellular cytotoxicity (ADCC), such as a neutrophil capable of inducingADCC. For example, monocytes, macrophages, which express FcR areinvolved in specific killing of target cells and presenting antigens toother components of the immune system, or binding to cells that presentantigens. In some embodiments, an effector cell may phagocytose a targetantigen, target cell, or microorganism. The expression of a particularFcR on an effector cell can be regulated by humoral factors such ascytokines. For example, expression of FcγRI has been found to beup-regulated by interferon γ (IFN-γ) and/or G-CSF. This enhancedexpression increases the cytotoxic activity of FcγRI-bearing cellsagainst targets. An effector cell can phagocytose or lyse a targetantigen or a target cell.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the presentinvention may include amino acid residues not encoded by human germlineimmunoglobulin sequences (for instance mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

As used herein, a human antibody is “derived from” a particular germlinesequence if the antibody is obtained from a system using humanimmunoglobulin sequences, for instance by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library, and wherein the selected human antibody isat least 90%, such as at least 95%, for instance at least 96%, such asat least 97%, for instance at least 98%, or such as at least 99%identical in amino acid sequence to the amino acid sequence encoded bythe germline VH or VL variable region gene segment. Typically, a humanantibody derived from a particular human germline VH or VL variableregion gene segment sequence will display no more than 10 amino aciddifferences, such as no more than 5, for instance no more than 4, 3, 2,or 1 amino acid difference from the amino acid sequence encoded by thegermline immunoglobulin gene.

A “chimeric” antibody is an antibody that contains one or more regionsfrom one antibody and one or more regions from one or more otherantibodies .derived from another species. A monovalent chimeric antibodyis a dimer (HL)) formed by a chimeric H chain associated throughdisulfide bridges with a chimeric L chain. A divalent chimeric antibodyis tetramer (H₂L₂) formed by two HL dimers associated through at leastone disulfide bridge. A polyvalent chimeric antibody may also beproduced, for example, by employing a CH region that oligomerizes (forinstance from an IgM H chain, or p chain). Typically, a chimericantibody refers to an antibody in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (see for instance U.S. Pat.No. 4,816,567 and Morrison et al., PNAS USA 81, 6851-6855 (1984)).Chimeric antibodies are produced by recombinant processes well known inthe art (see for instance Cabilly et al., PNAS USA 81, 3273-3277 (1984),Morrison et al., PNAS USA 81, 6851-6855 (1984), Boulianne et al., Nature312, 643-646 (1984), EP125023, Neuberger et al., Nature 314, 268-270(1985), EP171496, EP173494, WO86/01533, EP184187, Sahagan et al., J.Immunol. 137, 1066-1074 (1986), WO87/02671, Liu et al., PNAS USA 84,3439-3443 (1987), Sun et al., PNAS USA 84, 214-218 (1987), Better etal., Science 240, 1041-1043 (1988) and Harlow et al., Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., (1988)).

A “humanized” antibody is an antibody that is derived from a non-humanspecies, in which certain amino acids in the framework and constantdomains of the heavy and light chains have been mutated so as to avoidor abrogate an immune response in humans. Humanized forms of non-human(for instance murine) antibodies are chimeric antibodies which containminimal sequence derived from non-human immunoglobulin. For the mostpart, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a hypervariable region of the recipientare replaced by residues from a hypervariable region of a non-humanspecies (donor antibody) such as mouse, rat, rabbit or nonhuman primatehaving the desired antigen-binding characteristics such as specificity,and affinity. In some instances, Fv framework region (FR) residues ofthe human immunoglobulin are replaced by corresponding non-humanresidues. Furthermore, humanized antibodies may comprise residues whichare not found in the recipient antibody or in the donor antibody. Thesemodifications are made to further optimize antibody performance. Ingeneral, a humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. A humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321, 522-525 (1986), Riechmannet al., Nature 332, 323-329 (1988) and Presta, Curr. Op. Struct. Biol.2, 593-596 (1992).

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.Accordingly, the term “human monoclonal antibody” refers to antibodiesdisplaying a single binding specificity which have variable and constantregions derived from human germline immunoglobulin sequences. The humanmonoclonal antibodies may be generated by a hybridoma which includes a Bcell obtained from a transgenic or transchromosomal nonhuman animal,such as a transgenic mouse, having a genome comprising a human heavychain transgene and a light chain transgene, fused to an immortalizedcell. A monoclonal antibody may be abbreviated as mAb.

As used herein, “specific binding” refers to an antibody binding to apredetermined antigen. Typically, the antibody, binds with an affinitycorresponding to a K_(D) of about 10⁻⁷ M or less, such as about 10⁻⁸ Mor less, such as about 10⁻⁹ M or less, about 10⁻¹⁰ M or less, or about10⁻¹¹ M or even less when determined by surface plasmon resonance (SPR)technology in a BIAcore 3000 instrument using recombinant CD38 as theligand and the antibody as the analyte. The antibody may bind to thepredetermined antigen with an affinity corresponding to a K_(D) that isat least ten-fold lower, such as at least 100 fold lower, for instanceat least 1000 fold lower, such as at least 10,000 fold lower, forinstance at least 100,000 fold lower than its affinity for binding to anon-specific antigen (e.g., BSA, casein) other than the predeterminedantigen or a closely-related antigen. The amount with which the affinityis lower is dependent on the K_(D) of the antibody, so that when theK_(D) of the antibody is very low (that is, the antibody is highlyspecific), then the amount with which the affinity for the antigen islower than the affinity for a non-specific antigen may be at least10,000 fold.

The term specificity herein refers to the ability of a CD38 bindingpeptide, such as an anti-CD38 antibody, to recognize an epitope withinCD38, while only having little or no detectable reactivity with otherportions of CD38 (including other epitopes that are bound by otheranti-CD38 antibodies). Specificity can be relatively determined bycompetition assays as described herein. Specificity can moreparticularly be determined by any of the epitopeidentification/characterization techniques described herein or theirequivalents known in the art.

An antibody specific for a particular antigenic determinant maynonetheless cross-react with other biomolecules that may be present insome biological context with CD38. More typically, an anti-CD38antibody, may cross-react with CD38 homologues from other species. Ineither or both contexts, typically such cross-reactive antibodies areselective for human CD38 with respect to relevant structure and/orenvironmental factors.

The term “selectivity” herein refers to the preferential binding of ananti-CD38 antibody, for a particular region, target, or peptide;typically a region or epitope in CD38, as opposed to one or more otherbiological molecules, structures, cells, tissues, etc. In oneembodiment, an anti-CD38 antibody used in the present invention isselective for a portion of CD38 in the context of colon cancer cells(i.e., the anti-CD38 antibody will selectively bind to the portion ofCD38 over other components of a colon cancer cell).

The term “k_(d)” (sec⁻¹), as used herein, is intended to refer to thedissociation equilibrium rate constant of a particular antibody-antigeninteraction. Said value is also referred to as the k_(off) value.

The term “k_(a)” (M⁻¹×sec⁻¹), as used herein, is intended to refer tothe association equilibrium rate constant of a particularantibody-antigen interaction.

The term “K_(D)” (M), as used herein, is intended to refer to thedissociation equilibrium constant of a particular antibody-antigeninteraction.

The term “K_(A)” (M⁻¹), as used herein, is intended to refer to theassociation equilibrium constant of a particular antibody-antigeninteraction and is obtained by dividing the k_(a) by the k_(d).

As used herein, “isotype” refers to the antibody class (for instanceIgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is encoded by heavychain constant region genes.

“Target cell” shall mean any undesirable cell in an individual. In someembodiments, the target cell is a cell expressing or overexpressingCD38. Cells expressing CD38 typically include hemopoietic cells, such asmedullary thymocytes, activated T and B cells, 80% of resting NK cellsand monocytes, lymph node germinal center lymphoblasts, plasma B cellsand some intrafollicular cells, dendritic cells, normal bone marrowcells, particular precursor cells, 50-80% of umbilical cord blood cells,erythrocytes and platelets. CD38 can also be expressed bynon-hemopoietic cells, such as intra-epithelial cells and lamina proprialymphocytes in the gut, by Purkinje cells and neurofibrillary tangles inthe brain, by epithelial cells in the prostate, β-cells in the pancreas,osteoclasts in the bone, retinal cells in the eye, and sarcolemma ofsmooth and striated muscle. On malignant cells, CD38 is expressed in avariety of malignant hematological diseases, including but notrestricted to multiple myeloma, primary or secondary plasma cellleukemia, B-cell chronic lymphocytic leukemia, B-cell acute lymphocyticleukemia, Waldenström macroglobulinemia, primary systemic amyloidosis,mantle-cell lymphoma, pro-lymphocytic/myelocytic leukemia, acute myeloidleukemia, chronic myeloid leukemia, follicular lymphoma, and NK-cellleukemia.

As used herein, the term “individual” includes any human or non-humananimal. The term “non-human animal” includes all vertebrates, forinstance mammals and non-mammals, such as non-human primates, sheep,dog, cow, chickens, amphibians, reptiles, etc.

“Treatment” means the administration of an effective amount of atherapeutically active compound of the present invention with thepurpose of easing, ameliorating, or eradicating (curing) symptoms ordisease states.

Aspects and Embodiments of the Invention

In a first main aspect, the invention relates to a method for inhibitinggrowth and/or proliferation of tumor cells expressing CD38 in anindividual in need thereof, which method comprises administration to thesaid individual of

i) a non-agonistic antibody which binds to, i.e. binds specifically to,CD38,

ii) at least one corticosteroid, and

iii) at least one non-corticosteroid chemotherapeutic agent.

In a further main aspect, the invention relates to a method for treatingcancer involving tumor cells expressing CD38 in an individual in needthereof, which method comprises administration to the said individualof:

i) a non-agonistic antibody which binds to, i.e. binds specifically to,CD38,

ii) optionally at least one corticosteroid, and

iii) optionally at least one non-corticosteroid chemotherapeutic agent,such as a non-alkylating non-corticosteroid chemotherapeutic agent,

followed by autologous peripheral stem cell or bone marrowtransplantation.In one embodiment of the above methods of the invention, said at leastone non-corticosteroid chemotherapeutic agent comprises a cytotoxicagent and/or an angiogenesis inhibitor. In a further embodiment, said atleast one non-corticosteroid chemotherapeutic agent comprises analkylating agent.

In an even further embodiment, said at least one non-corticosteroidchemotherapeutic agent comprises one or more agents selected from thegroup consisting of: melphalan, mechlorethamine, thioepa, chlorambucil,carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan,dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine,mitomycin C, cisplatin and other platinum derivatives, such ascarboplatin.

In a further embodiment, said at least one non-corticosteroidchemotherapeutic agent comprises a glutamic acid derivative, such asthalidomide (Thalomid®) or a thalidomide analog, e.g. CC-5013(lenalidomide, Revlimid™) or CC4047 (Actimid™).

In an even further embodiment, said at least one non-corticosteroidchemotherapeutic agent comprises a proteasome inhibitor, such asbortezomib (Velcade®) or vinca alkaloid, such as vincristine or ananthracycline, such as doxorubicin.

In one embodiment of the methods of the invention, said at least onecorticosteroid comprises a glucocorticoid. In a further embodiment, saidat least one corticosteroid comprises prednisone or dexamethasone.In further embodiments of the invention, said at least onecorticosteroid comprises prednisone and said at least onenon-corticosteroid chemotherapeutic agent comprises melphalan.In even further embodiments of the invention, said at least onecorticosteroid comprises prednisone and said at least onenon-corticosteroid chemotherapeutic agent comprises thalidomide.In even further embodiments of the invention, said at least onecorticosteroid comprises prednisone and said at least onenon-corticosteroid chemotherapeutic agent comprises melphalan andthalidomide.In even further embodiments of the invention, said at least onecorticosteroid comprises dexamethasone and said at least onenon-corticosteroid chemotherapeutic agent comprises thalidomide and/orlenalidomide.In even further embodiments of the invention, said at least onecorticosteroid comprises dexamethasone and said at least onenon-corticosteroid chemotherapeutic agent comprises vincristine and/ordoxorubicin.In one embodiment of the methods of the invention, said non-agonisticantibody which binds to CD38 is a monoclonal antibody, such as a humanmonoclonal antibody.In a further embodiment of the invention, said antibody is an antagonistof CD38.In further embodiment of the invention, said antibody is:

an antibody that does not induce release of significant IL-6 by humanmonocytes or peripheral blood mononuclear cells as determined by themethod described in Example 19 of the specification

and/or

an antibody that does not induce release of detectable IFN-γ by human Tcells or peripheral blood mononuclear cells as determined by the methoddescribed in Example 20 of the specification

and/or

an antibody that is internalized by CD38 expressing cells; such asinternalized by CHO-CD38 cells within 5 to 15 minutes at 37° C. by themethod as described in Example 12 of the specification

and/or

an antibody that induces ADCC; such as with an EC₅₀ value of below 15ng/ml, such as below 10 ng/ml in Daudi-luc cells and with an EC₅₀ valueof below 75 ng/ml, such as below 50 ng/ml, 30 ng/ml or 10 ng/ml in MMcells as determined by the method described in Example 5 of thespecification

and/or

an antibody that induces CDC in the presence of complement; such as withan EC₅₀ value of below 5 μg/ml, such as below 1 μg/ml in daudi-luc orCD38-CHO cells by the method described in Example 6 of the specification

and/or

an antibody that inhibits the synthesis of cGDPR

and/or

an antibody that inhibits the synthesis of cADPR

and/or

an antibody that binds to human CD38 with an affinity (K_(D)) of below10⁻⁸ M, such as in the range of from 10⁻⁸ M to 10⁻¹¹ M, for example inthe range of from 7×10⁻⁹ M to 10⁻¹⁰ M, as determined by surface plasmonresonance as described in Example 20 of the specification

and/or

an antibody that inhibits the synthesis of cGDPR by at least 25%, suchas at least 30% after 90 minutes at a concentration of 3 μg/ml asdetermined by spectophotometric method described in Example 24 of thespecification

and/or

an antibody that inhibits the synthesis of cADPR by at least 25%, suchas at least 30% after 90 minutes at a concentration of 3 μg/ml asdetermined by the HPLC method described in Munshi et al., J. Biol. Chem.275, 21566-21571 (2000).

In one embodiment, the non-agonistic CD38 antibody used in the inventionis the antibody -003. -003 is a human monoclonal IgG1 antibody having aV_(L) region consisting of the sequence of SEQ ID No:2 and a V_(H)region consisting of the sequence of SEQ ID No:7.

In another embodiment, the non-agonistic CD38 antibody used in theinvention is the antibody -005. -005 is a human monoclonal IgG1 antibodyhaving a V_(L) region consisting of the sequence of SEQ ID No:12 and aV_(H) region consisting of the sequence of SEQ ID No:17.

In a further embodiment, the non-agonistic CD38 antibody used in theinvention is the antibody -024. -024 is a human monoclonal IgG1 antibodyhaving a V_(L) region consisting of the sequence of SEQ ID No:22 and aV_(H) region consisting of the sequence of SEQ ID No:27.

In one embodiment, the non-agonistic CD38 antibody used in the inventionis an antibody binding to human CD38 encoded by human light chain andhuman heavy chain nucleic acids comprising nucleotide sequences in theirvariable regions as set forth in SEQ ID No:1 and SEQ ID No:6,respectively.

In one embodiment, the non-agonistic CD38 antibody used in the inventionis an antibody binding to human CD38 encoded by human light chain andhuman heavy chain nucleic acids comprising nucleotide sequences in theirvariable regions as set forth in SEQ ID No:11 and SEQ ID No:16,respectively.

In one embodiment, the non-agonistic CD38 antibody used in the inventionis an antibody binding to human CD38 encoded by human light chain andhuman heavy chain nucleic acids comprising nucleotide sequences in theirvariable regions as set forth in SEQ ID No:21 and SEQ ID No:26,respectively.

In a yet further embodiment, the non-agonistic CD38 antibody used in theinvention is one of the antibodies described in WO2005/103083(Morphosys), in particular an antibody comprising one or more of thesequences given in FIG. 1b and/or the sequences given in FIG. 2B of WO2005/103083.

Antibodies interact with target antigens primarily through amino acidresidues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see for instance Riechmann, L. et al., Nature 332,323-327 (1998), Jones, P. et al., Nature 321, 522-525 (1986) and Queen,C. et al., PNAS USA 86, 10029-10033 (1989)).

Since it is well known in the art that antibody heavy chain CDR3 domainsplay a particularly important role in the binding specificity/affinityof an antibody for an antigen (Ditzel H J, et al., J Immunol. 157(2),739-49 (1996), Barbas S M et al., J. Am. Chem. Soc. 116, 2161-2162(1994), and Barbas S M et al., Proc Natl Acad Sci USA 92(7), 2529-33(1995), the antibodies used in the invention may comprise the heavychain CDR3s of -003 or -005 or -024. The antibodies used in theinvention may also comprise the heavy and light chain CDR3s of -003 or-005 or -024.

Thus, in a further embodiment of the methods of the invention, saidantibody is an antibody comprising a V_(H) CDR3 having the sequence asset forth in SEQ ID No:10 or an antibody which competes for CD38 bindingwith said antibody, e.g. by binding the same epitope as said antibody.

In one embodiment, the competition is determined by use of an ELISA asdescribed in the Examples section.

In another embodiment, the competition is determined by use of a FACS asdescribed in the Examples section.

In another embodiment, said antibody is an antibody comprising a V_(L)CDR3 having the sequence as set forth in SEQ ID No:5 and a V_(H) CDR3having the sequence as set forth in SEQ ID No:10.

In another embodiment, said antibody is an antibody comprising humanlight chain and human heavy variable regions, wherein the light chainvariable region comprises a V_(L) CDR1 having the sequence as set forthin SEQ ID No:3, a V_(L) CDR2 having the sequence as set forth in SEQ IDNo:4 and a V_(L) CDR3 having the sequence as set forth in SEQ ID No:5,and the heavy chain variable region comprises a V_(H) CDR1 having thesequence as set forth in SEQ ID No:8, a V_(H) CDR2 having the sequenceas set forth in SEQ ID No:9 and a V_(H) CDR3 having the sequence as setforth in SEQ ID No:10.

In another embodiment, said antibody is an antibody comprising a V_(L)region having the amino acid sequence as set forth in SEQ ID No:2 or aV_(L) region having at least about 90%, such as at least about 95% aminoacid sequence identity to the sequence as set forth in SEQ ID No:2.

In another embodiment, said antibody is an antibody comprising a V_(H)region having the amino acid sequence as set forth in SEQ ID No:7 or aV_(H) region having at least about 90%, such as at least about 95% aminoacid sequence identity to the sequence as set forth in SEQ ID No:7 or aV_(H) region having 1-5, such as 1-3 amino acid substitutions, deletionsor additions compared to the sequence as set forth in SEQ ID No:7.

In another embodiment, said antibody is an antibody comprising a V_(H)CDR3 having the sequence as set forth in SEQ ID No:20 or an antibodywhich competes for CD38 binding with said antibody, e.g. by binding thesame epitope as said antibody.

In another embodiment, said antibody is an antibody comprising a V_(L)CDR3 having the sequence as set forth in SEQ ID No:15 and a V_(H) CDR3having the sequence as set forth in SEQ ID No:20.

In another embodiment, said antibody is an antibody comprising humanlight chain and human heavy variable regions, wherein the light chainvariable region comprises a V_(L) CDR1 having the sequence as set forthin SEQ ID No:13, a V_(L) CDR2 having the sequence as set forth in SEQ IDNo:14 and a V_(L) CDR3 having the sequence as set forth in SEQ ID No:15,and the heavy chain variable region comprises a V_(H) CDR1 having thesequence as set forth in SEQ ID No:18, a V_(H) CDR2 having the sequenceas set forth in SEQ ID No:19 and a V_(H) CDR3 having the sequence as setforth in SEQ ID No:20.

In another embodiment, said antibody is an antibody comprising a V_(L)region having the amino acid sequence as set forth in SEQ ID No:12 or aV_(L) region having at least about 90%, such as at least about 95% aminoacid sequence identity to the sequence according to SEQ ID No:12.

In another embodiment, said antibody is an antibody comprising a V_(H)region having the amino acid sequence as set forth in SEQ ID No:17 or aV_(H) region having at least about 90%, such as at least about 95% aminoacid sequence identity to the sequence as set forth in SEQ ID No:17 or aV_(H) region having 1-5, such as 1-3 amino acid substitutions, deletionsor additions compared to the sequence as set forth in SEQ ID No:17.

In another embodiment, said antibody is an antibody comprising a V_(H)CDR3 having the sequence as set forth in SEQ ID No:30 or an antibodywhich competes for CD38 binding with said antibody, e.g. by binding thesame epitope as said antibody.

In another embodiment, said antibody is an antibody comprising a V_(L)CDR3 having the sequence as set forth in SEQ ID No:25 and a V_(H) CDR3having the sequence as set forth in SEQ ID No:30.

In another embodiment, said antibody is an antibody comprising humanlight chain and human heavy variable regions, wherein the light chainvariable region comprises a V_(L) CDR1 having the sequence as set forthin SEQ ID No:23, a V_(L) CDR2 having the sequence as set forth in SEQ IDNo:24 and a V_(L) CDR3 having the sequence as set forth in SEQ ID No:25,and the heavy chain variable region comprises a V_(H) CDR1 having thesequence as set forth in SEQ ID No:28, a V_(H) CDR2 having the sequenceas set forth in SEQ ID No:29 and a V_(H) CDR3 having the sequence as setforth in SEQ ID No:30.

In another embodiment, wherein said antibody is an antibody comprising aV_(L) region having the amino acid sequence as set forth in SEQ ID No:22or a V_(L) region having at least about 90%, such as at least about 95%amino acid sequence identity to the sequence according to SEQ ID No:22.

In another embodiment, said antibody is an antibody comprising a V_(H)region having the amino acid sequence as set forth in SEQ ID No:27 or aV_(H) region having at least about 90%, such as at least about 95% aminoacid sequence identity to the sequence according to SEQ ID No:27 or aV_(H) region having 1-5, such as 1-3 amino acid substitutions, deletionsor additions compared to the sequence as set forth in SEQ ID No:27.

In one embodiment of the methods of the invention, said at least onenon-corticosteroid chemotherapeutic agent comprises one or more agentsselected from the group consisting of: melphalan, mechlorethamine,thioepa, chlorambucil, carmustine (BSNU), lomustine (CCNU),cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine(DTIC), procarbazine, mitomycin C, cisplatin and other platinumderivatives, such as carboplatin, and said antibody is selected from thegroup consisting of:

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

In one embodiment of the methods of the invention, said at least onenon-corticosteroid chemotherapeutic agent comprises melphalan, and saidantibody is selected from the group consisting of:

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

In another embodiment, said at least one non-corticosteroidchemotherapeutic agent comprises a glutamic acid derivative, such asthalidomide (Thalomid®) or a thalidomide analog, e.g. CC-5013(lenalidomide, Revlimid™) or CC4047 (Actimid™), and said antibody isselected from the group consisting of:

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

In another embodiment, said at least one non-corticosteroidchemotherapeutic agent comprises a glutamic acid derivative, such asthalidomide (Thalomid®) or a thalidomide analog, e.g. CC-5013(lenalidomide, Revlimid™) or CC4047 (Actimid™), and said antibody is ahuman monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17.In another embodiment, said at least one non-corticosteroidchemotherapeutic agent comprises a proteasome inhibitor, such asbortezomib (Velcade®), and said antibody is selected from the groupconsisting of:

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

In another embodiment, said at least one corticosteroid comprisesdexamethasone, said at least one non-corticosteroid chemotherapeuticagent comprises a proteasome inhibitor, such as bortezomib (Velcade®),and said antibody is selected from the group consisting of:

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

In another embodiment, said at least one non-corticosteroidchemotherapeutic agent comprises a vinca alkaloid, such as vincristine,and said antibody is selected from the group consisting of:

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

In another embodiment, said at least one non-corticosteroidchemotherapeutic agent comprises an anthracycline, such as doxorubicin,and said antibody is selected from the group consisting of:

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

In another embodiment, said at least one corticosteroid comprises aglucocorticoid, and said antibody is selected from the group consistingof:

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

In another embodiment, said at least one corticosteroid comprisesprednisone, and said antibody is selected from the group consisting of:

human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

In another embodiment, said at least one corticosteroid comprisesprednisone and said at least one non-corticosteroid chemotherapeuticagent comprises melphalan, and said antibody is selected from the groupconsisting of:

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

In another embodiment, said at least one corticosteroid comprisesprednisone and said at least one non-corticosteroid chemotherapeuticagent comprises thalidomide, and said antibody is selected from thegroup consisting of:

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

In another embodiment, said at least one corticosteroid comprisesprednisone and said at least one non-corticosteroid chemotherapeuticagent comprises melphalan and thalidomide, and said antibody is selectedfrom the group consisting of:

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

In another embodiment, said at least one corticosteroid comprisesdexamethasone, and said antibody is selected from the group consistingof:

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

In another embodiment, said at least one corticosteroid comprisesdexamethasone and said at least one non-corticosteroid chemotherapeuticagent comprises thalidomide and/or lenalidomide, and said antibody isselected from the group consisting of:

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

In another embodiment, said at least one corticosteroid comprisesdexamethasone and said at least one non-corticosteroid chemotherapeuticagent comprises vincristine and/or doxorubicin, and said antibody isselected from the group consisting of:

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:2 and a V_(H) region consisting of the sequence ofSEQ ID No:7,

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:12 and a V_(H) region consisting of the sequenceof SEQ ID No:17, and

a human monoclonal IgG1 antibody having a V_(L) region consisting of thesequence of SEQ ID No:22 and a V_(H) region consisting of the sequenceof SEQ ID No:27.

Antibodies suitable for use in the present invention also includevariants of the antibodies of the Examples. A functional variant of aV_(L), V_(H), or CDR used in the context of a CD38 antibody still allowsthe antibody to retain at least a substantial proportion (at least about50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity/avidity and/orspecificity/selectivity of the parent antibody and in some cases such anantibody may be associated with greater affinity, selectivity, and/orspecificity than the parent antibody.

A “variant” anti-CD38 antibody is an antibody that differs from a parentantibody (typically generated by immunization) by one or more suitableamino acid residue alterations, that is substitutions, deletions,insertions, or terminal sequence additions, in the CDRs or other V_(H)and/or V_(L) sequences (provided that at least a substantial amount ofthe epitope binding characteristics of the parent antibody are retained,if not improved upon, by such changes).

Thus, for example, in an antibody variant one or more amino acidresidues may be introduced or inserted in or adjacent to one or more ofthe hypervariable regions of a parent antibody, such as in one or moreCDRs. An anti-CD38 antibody variant may comprise any number of insertedamino acid residues, provided again that at least a substantial amountof the epitope binding characteristics of the parent antibody areretained. An anti-CD38 antibody variant of the present invention may forexample comprise from about 1-30 inserted amino acid residues, forinstance from about 1-10, such as for instance from about 2-10, forinstance from 2-5 or such as from about 1-5 inserted amino acidresidues. Likewise, an anti-CD38 antibody variant of the presentinvention may for example comprise from about 1-30 deleted amino acidresidues, for instance from about 1-10, such as for instance from about2-10, for instance from 2-5 or such as from about 1-5 deleted amino acidresidues. Likewise, an anti-CD38 antibody variant of the presentinvention may for example comprise from about 1-30 substituted aminoacid residues, for instance from about 1-10, such as for instance fromabout 2-10, for instance from 2-5 or such as from about 1-5 substitutedamino acid residues. Likewise, an anti-CD38 antibody variant useful forthe present invention may for example comprise from about 1-30 terminalsequence amino acid residue additions, for instance from about 1-10,such as for instance from about 2-10, for instance from 2-5 or such asfrom about 1-5 terminal sequence amino acid residue additions. Aantibody variant of the present invention may also comprise acombination of two or more of such insertions, deletions, substitutionsand terminal sequence amino acid residue additions, provided that thevariant possesses at least a substantial proportion of the parentantibodies affinity, specificity, and/or selectivity with respect to oneor more CD38 epitopes.

In one embodiment, the antibody used in the invention comprises avariant V_(H) CDR3 consisting essentially of a sequence having at leastabout 80%, such as at least about 85%, for instance at least about 90%,such as at least about 95% amino acid sequence identity to a sequenceaccording to any one of SEQ ID No:10 or SEQ ID No:20 or SEQ ID No:30,wherein the antibody has at least a substantial proportion (at leastabout 50%, 60%, 70%, 80%, 90%, 95% or more) of the epitope bindingcharacteristics of an antibody having a variant V_(H) CDR3 sequence ofSEQ ID No:10 or SEQ ID No:20 or SEQ ID No:30, respectively, such as anantibody having a V_(H) sequence of SEQ ID No:7 or SEQ ID No:17 or SEQID No:27, respectively, such as an antibody having a V_(H) sequence ofSEQ ID No:7 and a V_(L) sequence of SEQ ID No:2, or an antibody having aV_(H) sequence of SEQ ID No:17 and a V_(L) sequence of SEQ ID No:12, oran antibody having a V_(H) sequence of SEQ ID No:27 and a V_(L) sequenceof SEQ ID No:22, respectively.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences may beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences may be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences may alsobe determined using the algorithm of E. Meyers and W. Miller, Comput.Appl. Biosci 4, 11-17 (1988)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences may be determined using the Needlemanand Wunsch, J. Mol. Biol. 48, 444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The sequence of CDR variants may differ from the sequence of the CDR ofthe parent antibody sequences through mostly conservative substitutions;for instance at least about 35%, about 50% or more, about 60% or more,about 70% or more, about 75% or more, about 80% or more, about 85% ormore, about 90% or more, about 95% or more (e.g., about 65-99%) of thesubstitutions in the variant are conservative amino acid residuereplacements. In the context of the present invention, conservativesubstitutions may be defined by substitutions within the classes ofamino acids reflected in one or more of the following three tables:

Amino Acid Residue Classes for Conservative Substitutions

Acidic Residues Asp and Glu Basic Residues Lys, Arg, and His HydrophilicUncharged Residues Ser, Thr, Asn, and Gln Aliphatic Uncharged ResiduesGly, Ala, Val, Leu, and Ile Non-polar Uncharged Residues Cys, Met, andPro Aromatic Residues Phe, Tyr, and Trp

Alternative Conservative Amino Acid Residue Substitution Classes

1 Ala (A) Ser (S) Thr (T) 2 Asp (D) Glu (E) 3 Asp (N) Gln (Q) 4 Arg (R)Lys (K) 5 Ile (I) Leu (L) Met (M) 6 Phe (F) Tyr (Y) Trp (W)

Alternative Physical and Functional Classifications of Amino AcidResidues

Alcohol group-containing residues S and T Aliphatic residues I, L, V,and M Cycloalkenyl-associated residues F, H, W, and Y Hydrophobicresidues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively chargedresidues D and E Polar residues C, D, E, H, K, N, Q, R, S, and TPositively charged residues H, K, and R Small residues A, C, D, G, N, P,S, T, and V Very small residues A, G, and S Residues involved in turnformation A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residues Q,T, K, S, G, P, D, E, and R

More conservative substitutions groupings include:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Additional groups of amino acids may also be formulated using theprinciples described in, e.g., Creighton (1984) Proteins: Structure andMolecular Properties (2d Ed. 1993), W.H. Freeman and Company.

Where hypervariable region insertions are made to generate a variantantibody, the typical range of lengths of the hypervariable region inquestion in known antibodies should be taken into consideration. Forexample, for the first hypervariable region of a light chain variabledomain, insertions may be introduced into the V_(L) CDR1 sequence of aparent antibody while retaining a substantially similar and therebyexpected appropriate size, which according to Kabat et al., supra, e.g.,typically has an overall of about 9-20 (e.g., about 10-17) residues.Similarly, V_(L) CDR2 typically has an overall length from about 5-10residues; V_(L) CDR3 typically has a length of about 7-20 residues;V_(H) CDR1 typically has a length of about 10-15 residues; V_(H) CDR2typically has a length of about 15-20 residues; and V_(H) CDR3 typicallyhas a length of about 6-30 residues (e.g., 3-25 residues). Insertions inthe V_(H) region typically are made in V_(H) CDR3 and typically near theC-terminal of the domain, such as about residues 97-102 of the parentV_(H) CDR3 (for instance adjacent to, or C-terminal in sequence to,residue number 100 of the parent V_(H) CDR3 sequence) using thealignment and numbering as described in Kabat. Antibody variants withinserted amino acid residue(s) in a hypervariable region thereof may beprepared randomly, especially where the starting binding affinity of theparent antibody for the target antigen is such that randomly producedantibody variants may be readily screened. For example, phage displayprovides a convenient method of screening such random variants.

In a further embodiment, the non-agonistic CD38 antibody used in theinvention is an antibody which is characterized with respect to itsability to compete (competitively inhibit) or cross-compete (i.e.,relatively partially inhibit epitope binding) with an antibody having aV_(L) sequence of SEQ ID No:2 and a V_(H) sequence of SEQ ID No:7 (suchas antibody -003), or an antibody having a V_(L) sequence of SEQ IDNo:12 and a V_(H) sequence of SEQ ID No:17 (such as antibody -005) or anantibody having a V_(L) sequence of SEQ ID No:22 and a V_(H) sequence ofSEQ ID No:27, (such as antibody -024), for binding to CD38. Such anantibody may be, for instance, a Fab fragment, derived from an antibodythat binds to an epitope identical to or overlapping with an epitopebound by an antibody having a V_(L) sequence of SEQ ID No:2 and a V_(H)sequence of SEQ ID No:7, or an antibody having a V_(L) sequence of SEQID No:12 and a V_(H) sequence of SEQ ID No:17 or an antibody having aV_(L) sequence of SEQ ID No:22 and a V_(H) sequence of SEQ ID No:27.Competition for binding to CD38 or a portion of CD38 by two or moreantibodies may be determined by any suitable technique. In oneembodiment, competition is determined for example as described inExample 7, 8 and 9.

Often competition is marked by a significantly greater relativeinhibition than about 5% as determined by ELISA and/or FACS analysis. Itmay be desirable to set a higher threshold of relative inhibition as acriteria/determinant of what is a suitable level of competition in aparticular context (e.g., where the competition analysis is used toselect or screen for new antibodies designed with the intended functionof blocking the binding of another peptide or molecule binding to CD38(e.g., the natural binding partners of CD38 such as CD31, also calledCD31 antigen, EndoCAM, GPIIA′, PECAM-1, platelet/endothelial celladhesion molecule or naturally occurring anti-CD38 antibody)). Thus, forexample, it is possible to set a criteria for competitiveness wherein atleast about 10% relative inhibition is detected; at least about 15%relative inhibition is detected; or at least about 20% relativeinhibition is detected before an antibody is considered sufficientlycompetitive. In cases where epitopes belonging to competing antibodiesare closely located in an antigen, competition may be marked by greaterthan about 40% relative inhibition of CD38 binding (e.g., at least about45% inhibition, such as at least about 50% inhibition, for instance atleast about 55% inhibition, such as at least about 60% inhibition, forinstance at least about 65% inhibition, such as at least about 70%inhibition, for instance at least about 75% inhibition, such as at leastabout 80% inhibition, for instance at least about 85% inhibition, suchas at least about 90% inhibition, for instance at least about 95%inhibition, or higher level of relative inhibition).

In a further embodiment, the non-agonistic CD38 antibody used in theinvention is an antibody that specifically binds to a CD38 epitope thatalso is specifically bound by an antibody having a V_(L) sequence of SEQID No:2 and a V_(H) sequence of SEQ ID No:7 (such as antibody -003), oran antibody having a V_(L) sequence of SEQ ID No:12 and a V_(H) sequenceof SEQ ID No:17 (such as antibody -005) or an antibody having a V_(L)sequence of SEQ ID No:22 and a V_(H) sequence of SEQ ID No:27 (such asantibody -024).

A CD38 epitope bound by an antibody having a V_(L) sequence of SEQ IDNo:2 and a V_(H) sequence of SEQ ID No:7 (such as the antibody -003), oran antibody having a V_(L) sequence of SEQ ID No:12 and a V_(H) sequenceof SEQ ID No:17 (such as the antibody -005) or an antibody having aV_(L) sequence of SEQ ID No:22 and a V_(H) sequence of SEQ ID No:27(such as antibody -024), may be identified via standard mapping andcharacterization techniques, further refinement of which may beidentified by any suitable technique, numerous examples of which areavailable to the skilled artisan. These techniques may also be used toidentify and/or characterize epitopes for anti-CD38 antibodiesgenerally. As one example of such mapping/characterization methods, anepitope for an anti-CD38 antibody may be determined by epitope“foot-printing” using chemical modification of the exposedamines/carboxyls in the CD38 protein. One specific example of such afoot-printing technique is the use of HXMS (hydrogen-deuterium exchangedetected by mass spectrometry) wherein a hydrogen/deuterium exchange ofreceptor and ligand protein amide protons, binding, and back exchangeoccurs, wherein the backbone amide groups participating in proteinbinding are protected from back exchange and therefore will remaindeuterated. Relevant regions may be identified at this point by pepticproteolysis, fast microbore high-performance liquid chromatographyseparation, and/or electrospray ionization mass spectrometry. See, e.g.,Ehring H, Analytical Biochemistry, 267(2) 252-259 (1999) and/or Engen,J. R. and Smith, D. L. (2001) Anal. Chem. 73, 256A-265A. Another exampleof a suitable epitope identification technique is nuclear magneticresonance epitope mapping (NMR), where typically the position of thesignals in two-dimensional NMR spectres of the free antigen and theantigen complexed with the antigen binding peptide, such as an antibody,are compared. The antigen typically is selectively isotopically labeledwith ¹⁵N so that only signals corresponding to the antigen and nosignals from the antigen binding peptide are seen in the NMR-spectrum.Antigen signals originating from amino acids involved in the interactionwith the antigen binding peptide typically will shift position in thespectres of the complex compared to the spectres of the free antigen,and the amino acids involved in the binding may be identified that way.See for instance Ernst Schering Res Found Workshop. (44), 149-67 (2004),Huang et al., Journal of Molecular Biology 281(1), 61-67 (1998) andSaito and Patterson, Methods. 9(3), 516-24 (1996).

Epitope mapping/characterization may also be performed using massspectrometry methods. See for instance Downward, J Mass Spectrom. 35(4),493-503 (2000) and Kiselar and Downard, Anal Chem. 71(9), 1792-801(1999).

Protease digestion techniques may also be useful in the context ofepitope mapping and identification. Antigenic determinant-relevantregions/sequences may be determined by protease digestion, e.g. by usingtrypsin in a ratio of about 1:50 to CD38 overnight (O/N) digestion at37° C. and pH 7-8, followed by mass spectrometry (MS) analysis forpeptide identification. The peptides protected from trypsin cleavage bythe antibody may subsequently be identified by comparison of samplessubjected to trypsin digestion and samples incubated with antibody andthen subjected to digestion by e.g. trypsin (thereby revealing a footprint for the binder). Other enzymes like chymotrypsin, pepsin, etc. mayalso or alternatively be used in a similar epitope characterizationmethod. An antibody which gives the significantly same result as anantibody having a V_(L) sequence of SEQ ID No:2 and a V_(H) sequence ofSEQ ID No:7 (such as the antibody -003), or an antibody having a V_(L)sequence of SEQ ID No:12 and a V_(H) sequence of SEQ ID No:17 (such asthe antibody -005) or an antibody having a V_(L) sequence of SEQ IDNo:22 and a V_(H) sequence of SEQ ID No:27 (such as antibody -024) inthese measurements are deemed to be an antibody that bind the sameepitope as an antibody having a V_(L) sequence of SEQ ID No:2 and aV_(H) sequence of SEQ ID No:7 (such as the antibody -003), or anantibody having a V_(L) sequence of SEQ ID No:12 and a V_(H) sequence ofSEQ ID No:17 (such as the antibody -005) or an antibody having a V_(L)sequence of SEQ ID No:22 and a V_(H) sequence of SEQ ID No:27 (such asantibody -024), respectively. See for instance Manca, Ann Ist SuperSanita. 27(1), 15-9 (1991) for a discussion of similar techniques.

Other methods potentially helpful in mapping epitopes includecrystallography techniques, X-ray diffraction techniques (such as theX-ray diffraction/sequence study techniques developed by Poljak andothers in the 1970s-1980s), and the application of Multipin PeptideSynthesis Technology. Computer-based methods such as sequence analysisand three dimensional structure analysis and docking may also be used toidentify antigenic determinants. For example, an epitope may also bedetermined by molecular modeling using a structure of CD38 with dockingof the structure of the Fab fragment of the individual monoclonalantibody. These and other mapping methods are discussed in EpitopeMapping A Practical Approach (Westwood and Hay Eds.) 2001 OxfordUniversity Press.

An antibody used in the present invention may have any suitable affinityand/or avidity for one or more epitopes contained at least partially inCD38. Affinity refers to the strength of binding of the antibody to suchan epitope. Typically, affinity is measured by dissociation constantK_(d), defined as [Ab]×[Ag]/[Ab−Ag] where [Ab−Ag] is the molarconcentration of the antibody-antigen complex (or the antibody-antigencomplex), [Ab] is the molar concentration of the unbound antibody and[Ag] is the molar concentration of the unbound antigen. The affinityconstant K_(a) is defined by 1/K_(d). Suitable methods for determiningspecificity and affinity by competitive inhibition can be found in forinstance Harlow et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Colligan etal., eds., Current Protocols in Immunology, Greene Publishing Assoc. andWiley InterScience N.Y., (1992, 1993) and Muller, Meth. Enzymol. 92,589-601 (1983).

Anti-CD38 antibodies used in the present invention may have an affinityfor at least one epitope at least partially comprised in CD38 in therange of about 10⁴ to about 10¹⁰ M⁻¹. Such an antibody may have anaffinity that is at least as great for CD38 as -003 and -005 and -024,and in some embodiments have an affinity that is at least about as greatas -003 and -005 and -024. Affinity may be determined by any of themethods described elsewhere herein or their known equivalents in theart. An example of one method that may be used to determine affinity isprovided in Scatchard analysis of Munson & Pollard, Anal. Biochem. 107,220 (1980). Binding affinity also may be determined by equilibriummethods (for instance enzyme-linked immunoabsorbent assay (ELISA) orradioimmunoassay (RIA)) or kinetics analysis (for instance BIACORE™analysis).

Typically, the disassociation constant for anti-CD38 antibodies used inthe present invention is less than about 100 nM, less than about 50 nM,less than about 10 nM, about 5 nM or less, about 1 nM or less, about 0.5nM or less, about 0.1 nM or less, about 0.01 nM or less, or even about0.001 nM or less.

Non-limiting examples of anti-CD38 antibodies suitable for use in thepresent invention include (a) a complete functional, immunoglobulinmolecule comprising: (i) two identical chimeric heavy chains comprisinga variable region with a human B cell surface antigen specificity andhuman constant region and (ii) two identical all (i.e. non-chimeric)human light chains; (b) a complete, functional, immunoglobulin moleculecomprising: (i) two identical chimeric heavy chains comprising avariable region as indicated, and a human constant region, and (ii) twoidentical all (i.e. non-chimeric) non-human light chains; (c) amonovalent antibody, i.e., a complete, functional immunoglobulinmolecule comprising: (i) two identical chimeric heavy chains comprisinga variable region as indicated, and a human constant region, and (ii)two different light chains, only one of which has the same specificityas the variable region of the heavy chains. The resulting antibodymolecule binds only to one end thereof and is therefore incapable ofdivalent binding. As another illustration, immunoglobulin-relatedpeptides provided by the present invention may be said to include thefollowing: (a) a whole immunoglobulin molecule; (b) an scFv; (c) amonoclonal antibody; (d) a human antibody; (e) a chimeric antibody; (f)a humanized antibody; (g) a Fab fragment; (h) an Fab′ fragment; (i) anF(ab′)₂ fragment; (j) an Fv molecule; and (k) a disulfide-linked Fvmolecule.

In one embodiment, the antibody used in the present invention is apolyclonal antibody. In one embodiment, the antibody used in of thepresent invention is an monoclonal antibody. In a further embodiment,the antibody used in of the present invention is a human monoclonalantibody. In another further embodiment, the antibody used in of thepresent invention is a humanized antibody. In another furtherembodiment, the antibody used in of the present invention is a chimericantibody. In another further embodiment, the antibody used in of thepresent invention is a monoclonal antibody originating entirely from amammalian species different from humans. In a further embodiment, theantibody used in of the present invention is a fully murine monoclonalantibody.

In one embodiment, the antibody used in the invention is glycosylated ina eukaryotic cell. In another embodiment, the antibody used in theinvention further comprises a chelator linker for attaching aradioisotope. In a further embodiment, the antibody used in theinvention is in a substantially isolated form.

A monoclonal antibody refers to a composition comprising a homogeneousantibody population having a uniform structure and specificity.Typically a monoclonal antibody is an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific and each monoclonal antibodyis typically directed against a single epitope, which is in contrast topolyclonal antibody preparations which typically include differentantibodies directed against different epitopes. That an antibody ismonoclonal is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies of the present invention may be produced by the hybridomamethod first described by Kohler et al., Nature 256, 495 (1975), or maybe produced by recombinant DNA methods. Monoclonal antibodies may alsobe isolated from phage antibody libraries using the techniques describedin, for example, Clackson et al., Nature 352, 624-628 (1991) and Markset al., J. Mol. Biol. 222, 581-597 (1991).

Monoclonal antibodies may be obtained from any suitable source. Thus,for example, monoclonal antibodies may be obtained from hybridomasprepared from murine splenic B cells obtained from mice immunized withan antigen of interest, for instance in form of cells expressing theantigen on the surface, or a nucleic acid encoding an antigen ofinterest. Monoclonal antibodies may also be obtained from hybridomasderived from antibody-expressing cells of immunized humans or non-humanmammals such as rats, dogs, primates, etc.

Alternatively, the cloned antibody genes can be expressed in otherexpression systems, including prokaryotic cells, such as microorganisms,such as E. coli, for the production of single chain Fv antibodies, algi,as well as insect cells. Furthermore, the antibodies can be produced intransgenic non-human animals, such as in milk from sheep and rabbits orin eggs from hens, or in transgenic plants. See for instance Verma, R.,et al., J. Immunol. Meth. 216, 165-181 (1998); Pollock, et al., J.Immunol. Meth. 231, 147-157 (1999); and Fischer, R., et al., Biol. Chem.380, 825-839 (1999).

In one embodiment, human monoclonal antibodies directed against CD38 maybe generated using transgenic or transchromosomal mice carrying parts ofthe human immune system rather than the mouse system. Such transgenicand transchromosomic mice include mice referred to herein as HuMAb miceand KM mice, respectively, and are collectively referred to herein as“transgenic mice”. A human monoclonal antibody generated in such micemay be abbreviated as HuMab.

The HuMAb mouse contains a human immunoglobulin gene miniloci thatencodes unrearranged human heavy (μ and γ) and κ light chainimmunoglobulin sequences, together with targeted mutations thatinactivate the endogenous p and K chain loci (Lonberg, N. et al., Nature368, 856-859 (1994)). Accordingly, the mice exhibit reduced expressionof mouse IgM or κ and in response to immunization, the introduced humanheavy and light chain transgenes, undergo class switching and somaticmutation to generate high affinity human IgG,κ monoclonal antibodies(Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook ofExperimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar,D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. andLonberg, N. Ann. N.Y. Acad. Sci 764 536-546 (1995)). The preparation ofHuMAb mice is described in detail in Taylor, L. et al., Nucleic AcidsResearch 20, 6287-6295 (1992), Chen, J. et al., International Immunology5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994),Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild,D. et al., Nature Biotechnology 14, 845-851 (1996). See also U.S. Pat.Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397,5,661,016, 5,814,318, 5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.

The HCo7 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCo5 human kappa light chain transgene (asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)),and a HCo7 human heavy chain transgene (as described in U.S. Pat. No.5,770,429).

The HCo12 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCo5 human kappa light chain transgene (asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)),and a HCo1 2 human heavy chain transgene (as described in Example 2 ofWO 01/14424). In the KM mouse strain, the endogenous mouse kappa lightchain gene has been homozygously disrupted as described in Chen et al.,EMBO J. 12, 811-820 (1993) and the endogenous mouse heavy chain gene hasbeen homozygously disrupted as described in Example 1 of WO 01/09187.This mouse strain carries a human kappa light chain transgene, KCo5, asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).This mouse strain also carries a human heavy chain transchromosomecomposed of chromosome 14 fragment hCF (SC20) as described in WO02/43478.

The KM mouse contains a human heavy chain transchromosome and a humankappa light chain transgene. The endogenous mouse heavy and light chaingenes also have been disrupted in the KM mice such that immunization ofthe mice leads to production of human immunoglobulins rather than mouseimmunoglobulins. Construction of KM mice and their use to raise humanimmunoglobulins is described in detail in WO 02/43478.Splenocytes fromthese transgenic mice may be used to generate hybridomas that secretehuman monoclonal antibodies according to well known techniques.

Human monoclonal or polyclonal antibodies used in the present invention,or antibodies used in the present invention originating from otherspecies may also be generated transgenically through the generation ofanother non-human mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies may beproduced in, and recovered from, the milk of goats, cows, or othermammals. See for instance U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172and 5,741,957.

Further, human antibodies used in the present invention or antibodiesused in the present invention from other species may be generatedthrough display-type technologies, including, without limitation, phagedisplay, retroviral display, ribosomal display, and other techniques,using techniques well known in the art and the resulting molecules maybe subjected to additional maturation, such as affinity maturation, assuch techniques are well known in the art (see for instance Hoogenboomet al., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al.,Nature Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNASUSA 94, 4937-4942 (1997) (ribosomal display), Parmley and Smith, Gene73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992),Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russel et al., Nucl. AcidsResearch 21, 1081-1085 (1993), Hoogenboom et al., Immunol. Reviews 130,43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84 (1992), and U.S.Pat. No. 5,733,743). If display technologies are utilized to produceantibodies that are not human, such antibodies may be humanized, forinstance as described elsewhere herein.

Examples of how to make humanized antibodies may be found in forinstance U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293. Also, theuse of Ig cDNA for construction of chimeric immunoglobulin genes isknown in the art (see for instance Liu et al., PNAS USA 84, 3439 (1987)and J. Immunol. 139, 3521 (1987

Anti-CD38 antibodies may be recovered from recombinant combinatorialantibody libraries, such as a scFv phage display library, which may bemade with human V_(L) and V_(H) cDNAs prepared from mRNA derived fromhuman lymphocytes. Methods for preparing and screening such librariesare known in the art. There are a number of commercially available kitsfor generating phage display libraries. There are also other methods andreagents that may be used in generating and screening antibody displaylibraries (see for instance U.S. Pat. No. 5,223,409, WO 92/18619, WO91/17271, WO 92/20791, WO 92/15679, WO 93/01288, WO 92/01047, WO92/09690, Fuchs et al., Bio/Technology 9, 1370-1372 (1991), Hay et al.,Hum. Antibod. Hybridomas 3, 81-85 (1992), Huse et al., Science 246,1275-1281 (1989), McCafferty et al., Nature 348, 552-554 (1990),Griffiths et al., EMBO J 12, 725-734 (1993), Hawkins et al., J. Mol.Biol. 226, 889-896 (1992), Clackson et al., Nature 352, 624-628 (1991),Gram et al., PNAS USA 89, 3576-3580 (1992), Garrad et al.,Bio/Technology 9, 1373-1377 (1991), Hoogenboom et al., Nuc Acid Res 19,4133-4137 (1991) and Barbas et al., PNAS USA 88, 7978-7982 (1991)).Suitable V_(L) and V_(H) nucleic acid sequences may be selected usingany appropriate method. For example, V_(L) and V_(H) nucleic acids maybe selected by employing the epitope imprinting methods described in WO93/06213. Antibody libraries, such as scFv libraries may be prepared andscreened using known and suitable methods (with human CD38-containingpeptides as antigen(s)), such as those described in for instanceWO92/01047, McCafferty et al., Nature 348, 552-554 (1990) and Griffithset al., EMBO J 12, 725-734 (1993). Such antibody libraries are featuresof the present invention that may be used therapeutically to provide amore comprehensive immune response; as tools in screening methods forimmunogenic peptides, small molecules, other anti-CD38 antibodies (e.g.,by way of competition assays), and the like; and/or in diagnosticmethods and compositions (e.g., an immunoassay chip comprising a panelof such antibodies optionally in association with other antibodies maybe prepared by standard techniques). Once initial human V_(L) and V_(H)segments are selected, “mix and match” experiments, in which differentpairs of the initially selected V_(L) and V_(H) segments are screenedfor CD38-containing peptide binding, may be performed to selectdesirable V_(L)/V_(H) pair combinations. For example, reactivity of thepeptides may be determined by ELISA or other suitable epitope analysismethods (see for instance Scott, J. K. and Smith, G. P. Science 249,386-390 (1990), Cwirla et al., PNAS USA 87, 6378-6382 (1990), Felici etal., J. Mol. Biol. 222, 301-310 (1991) and Kuwabara et al., NatureBiotechnology 15, 74-78 (1997) for discussion of such techniques andprinciples). Antibodies may be selected by their affinity for antigenand/or by their kinetics of dissociation (off-rate) from antigen (seefor instance Hawkins et al., J. Mol. Biol. 226, 889-896 (1992)).

High-affinity antibody peptides, such as human single-chain Fv (scFv)and Fab antibody fragments, may also be isolated from such librariesusing a panning technique in which the antigen of interest isimmobilized on a solid surface, such as microtiter plates or beads (seefor instance Barbas and Burton, Trends. Biotechnol. 14, 230-234 (1996)and Aujame et al., Hum. Antibodies 8, 155-68 (1997). Phage display oflarge naïve libraries also makes it possible to isolate human antibodiesdirectly without immunization (see for instance de Haard et al., J.Biol. Chem. 274(26), 18218-18230 (1999)).

Antibodies suitable for use in the present invention may be selectedbased on their ability to provide the ability of complement fixation, ornot. There are a number of isotypes of antibodies that are capable ofcomplement fixation and CDC, including, without limitation, thefollowing: murine IgM, murine IgG2a, murine IgG2b, murine IgG3, humanIgM, human IgG1, and human IgG3. Those isotypes that do not include,without limitation, human IgG2 and human IgG4. Isotype determination andother methods for modifying the complement fixation and CDC functionalcharacteristics of antibodies are known in the art.

Anti-CD38 antibodies used in the present invention may be prepared byrecombinant expression in any suitable type of cells or animals.Suitable methods for antibody production are known in the art andinclude those described in for instance Harlow et al., Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., (1988), Harlow and Lane: Using Antibodies: A LaboratoryManual (Cold Spring Harbor Laboratory Press (1999)), U.S. Pat. No.4,376,110 and Ausubel et al., eds., Current Protocols In MolecularBiology, Greene Publishing Assoc. and Wiley InterScience N.Y., (1987,1992). Monoclonal antibodies may be made using the hybridoma methodfirst described by Kohler et al., Nature 256, 495 (1975), or by otherwell-known, subsequently-developed methods (see, e.g., Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)). Hybridomas useful in the production of anti-CD38antibodies of the present invention are also provided by the presentinvention. Such hybridomas may be formed by chemical fusion, electricalfusion, or any other suitable technique, with any suitable type ofmyeloma, heteromyeloma, phoblastoid cell, plasmacytoma or otherequivalent thereof and any suitable type of antibody-expressing cell.Transformed immortalized B cells may also be used to efficiently produceantibodies of the present invention and are also provided by the presentinvention. Such cells may be produced by standard techniques, such astransformation with an Epstein Barr Virus, or a transforming gene. (See,e.g., “Continuously Proliferating Human Cell Lines Synthesizing Antibodyof Predetermined Specificity,” Zurawaki, V. R. et al., in MonoclonalAntibodies, ed. by Kennett R. H. et al., Plenum Press, N.Y. 1980, pp19-33.).

Recombinant cells comprising exogenous nucleic acids encoding anti-CD38antibodies may be prepared by any suitable technique (e.g.,transfection/transformation with a naked DNA plasmid vector, viralvector, invasive bacterial cell vector or other whole cell vector, etc.,comprising a antibody-encoding sequence (or sequences) delivered intothe cell by calcium phosphate-precipitation facilitated transfection,receptor-mediated targeting and transfection, biolistic delivery,electroporation, dextran-mediated transfection, liposome-mediatedtransformation, protoplast fusion, direct microinjection, etc.). Methodsof transforming/transfecting cells are well known in the art (see, e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press (2d Edition, 1989 and 3rd Edition, 2001) and F.Ausubel et al., ed. Current Protocols in Molecular Biology, GreenePublishing and Wiley InterScience New York (1987). Such recombinantcells are a feature of the present invention.

Cell lines available as hosts for recombinant protein expression arewell known in the art and include many immortalized cell lines availablefrom the American Type Culture Collection (ATCC). These include, interalia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells,baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a numberof other cell lines. Other cell lines that may be used are insect celllines, such as Sf9 cells. When nucleic acids (or nucleic acid-containingvectors) encoding proteins, such as anti-CD38 antibodies), areintroduced into mammalian host cells, proteins may be produced byculturing the host cells for a period of time sufficient to allow forexpression of the protein in the host cells or by secretion of theprotein into the culture medium in which the host cells are grown.Antibodies may be recovered from the culture medium using standardprotein purification methods. Antibodies may also be recovered from hostcell lysates when directly expressed without a secretory signal.

When recombinant expression vectors encoding anti-CD38 antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or for secretion of theantibody into the culture medium in which the host cells are grown. Thepurification of antibodies from cell cultures, cell lysates, and animals(e.g., from the ascites fluid of a transgenic animal producing anti-CD38antibodies) may be achieved by application of any number of suitabletechniques known in the art including, e.g., immunoaffinity columnpurification; sulfate precipitation; chromatofocusing; preparativeSDS-PAGE, and the like.

Human monoclonal antibodies of the present invention may also beproduced by a variety of other techniques, including conventionalmonoclonal antibody methodology, e.g., the standard somatic cellhybridization technique of Kohler and Milstein, Nature 256, 495 (1975).Other techniques for producing monoclonal antibody may also be employed,e.g. phage display techniques using libraries of human antibody genes.In one embodiment, anti-CD38 antibodies of the present inventionproduced by use of hybridomas generated in a murine system. Hybridomaproduction in the mouse is a very well established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

To generate fully human monoclonal antibodies to CD38, transgenic ortranschromosomal mice containing human immunoglobulin genes (e.g.,HCo12, HCo7 or KM mice) may be immunized with an enriched preparation ofCD38 antigen and/or cells expressing CD38, as described, for example, byLonberg et al., (1994), supra, Fishwild et al., (1996), supra, and WO98/24884. Alternatively, mice may be immunized with DNA encoding humanCD38. The mice may be 6-16 weeks of age upon the first infusion. Forexample, an enriched preparation (5-50 μg) of the CD38 antigen may beused to immunize the HuMAb mice intraperitoneally. In the event thatimmunizations using a purified or enriched preparation of the CD38antigen do not result in antibodies, mice may also be immunized withcells expressing CD38, e.g., a cell line, to promote immune responses.

Cumulative experience with various antigens has shown that the HuMAbtransgenic mice respond best when initially immunized intraperitoneally(i.p.) or subcutaneously (s.c.) with CD38 expressing cells in completeFreund's adjuvant, followed by every other week i.p. immunizations (upto a total of 10) with CD38 expressing cells in PBS. The immune responsemay be monitored over the course of the immunization protocol withplasma samples being obtained by retroorbital bleeds. The plasma may bescreened by FACS analysis, and mice with sufficient titers of anti-CD38human immunoglobulin may be used for fusions. Mice may be boostedintravenously with CD38 expressing cells for Examples 4 and 3 daysbefore sacrifice and removal of the spleen.

To generate hybridomas producing human monoclonal antibodies to humanCD38, splenocytes and lymph node cells from immunized mice may beisolated and fused to an appropriate immortalized cell line, such as amouse myeloma cell line. The resulting hybridomas may then be screenedfor the production of antigen-specific antibodies. For example, singlecell suspensions of splenic lymphocytes from immunized mice may be fusedto SP2/0 nonsecreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG(w/v). Cells may be plated at approximately 1×105 per well in flatbottom microtiter plate, followed by a two week incubation in selectivemedium containing besides usual reagents 10% fetal Clone Serum, 5-10%origen hybridoma cloning factor (IGEN) and 1× HAT (Sigma). Afterapproximately two weeks, cells may be cultured in medium in which theHAT is replaced with HT. Individual wells may then be screened by ELISAfor human kappa-light chain containing antibodies and by FACS analysisusing CD38 expressing cells for CD38 specificity. Once extensivehybridoma growth occurs, medium may be observed usually after 10-14days. The antibody secreting hybridomas may be replated, screened again,and if still positive for human IgG, anti-CD38 monoclonal antibodies maybe subcloned at least twice by limiting dilution. The stable subclonesmay then be cultured in vitro to generate antibody in tissue culturemedium for characterization.

Human antibodies of the present invention may also be produced in a hostcell transfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art,see for instance Morrison, S., Science 229, 1202 (1985).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, may beobtained by standard molecular biology techniques (for instance PCRamplification, site directed mutagenesis) and may be inserted intoexpression vectors such that the genes are operatively linked totranscriptional and translational control sequences. In this context,the term “operatively linked” is intended to mean that an antibody geneis ligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene may be inserted into separatevectors or, more typically, both genes are inserted into the sameexpression vector. The antibody genes may be inserted into theexpression vector by standard methods (e.g., ligation of complementaryrestriction sites on the antibody gene fragment and vector, or blunt endligation if no restriction sites are present). The light and heavy chainvariable regions of the antibodies described herein may be used tocreate full-length antibody genes of any antibody isotype by insertingthem into expression vectors already encoding heavy chain constant andlight chain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the CH segment(s) within the vector andthe V_(L) segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectormay encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene may be cloned into thevector such that the signal peptide is linked in-frame to the aminoterminus of the antibody chain gene. The signal peptide may be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors carry regulatory sequences that allows and control theexpression of the antibody chain genes in a host cell. Furthermore, therecombinant expression vectors may carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. The selectablemarker gene facilitates selection of host cells into which the vectorhas been introduced (see for instance U.S. Pat. Nos. 4,399,216,4,634,665 and 5,179,017). For example, typically the selectable markergene confers resistance to drugs, such as G418, hygromycin ormethotrexate, on a host cell into which the vector has been introduced.Examples of selectable marker genes include the dihydrofolate reductase(DHFR) gene (for use in dhfr-host cells with methotrexateselection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The host cells may be prokaryotic or eukaryotic,such as mammalian, host cells. For instance antigen binding fragmentsmay be expressed in prokaryotic host cells and full-length antibodiesmay be expressed in eukaryotic host cells.

In one embodiment the antibodies are expressed in eukaryotic cells, suchas mammalian host cells. Examples of mammalian host cells for expressingthe recombinant antibodies of the present invention include CHO cells(including dhfr-CHO cells, described in Urlaub and Chasin, PNAS USA 77,4216-4220 (1980), used with a DHFR selectable marker, for instance asdescribed in R. J. Kaufman and P. A. Sharp, Mol. Biol. 159, 601-621(1982)), NS/0 myeloma cells, COS cells, HEK293 cells and SP2.0 cells. Inparticular for use with NS/0 myeloma cells, another example of aexpression system is the GS (glutamine synthetase) gene expressionsystem disclosed in WO87/04462, WO89/01036 and EP338 841.

The antibody genes may be expressed in other expression systems,including prokaryotic cells, such as microorganisms, e.g. E. coli forthe production of scFv antibodies, algi, as well as insect cells.Furthermore, the antibodies may be produced in transgenic non-humananimals, such as in milk from sheep and rabbits or eggs from hens, or intransgenic plants. See for instance Verma, R. et al., J. Immunol. Meth.216, 165-181 (1998), Pollock et al., J. Immunol. Meth. 231, 147-157(1999) and Fischer, R. et al., Biol. Chem. 380, 825-839 (1999).

Bispecific and Multispecific Antibodies

In one embodiment of the present invention, the antibody used may bederivatized or linked to another functional molecule, for instanceanother peptide or protein (such as a Fab′ fragment) to generate abispecific or multispecific molecule which binds to multiple bindingsites or target epitopes. For example, an antibody used in the presentinvention may be functionally linked (for instance by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother binding molecules, such as another antibody, peptide or bindingmimetic.

Accordingly, the present invention includes the use of bispecific andmultispecific molecules comprising at least one first bindingspecificity for CD38 and a second binding specificity for a secondtarget epitope. In one embodiment of the present invention, the secondtarget epitope is an Fc receptor, e.g., human FcγRI (CD64) or a humanFcα receptor (CD89), or a T cell receptor, e.g., CD3. In one embodiment,the present invention provides bispecific and multispecific moleculescapable of binding both to FcγR, FcαR or FcεR expressing effector cells(e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and totarget cells expressing CD38. These bispecific and multispecificmolecules target CD38 expressing cells to effector cell and trigger Fcreceptor-mediated effector cell activities, such as phagocytosis of CD38expressing cells, antibody dependent cellular cytotoxicity (ADCC),cytokine release, or generation of superoxide anion.

In one embodiment, the bispecific and multispecific molecules used inthe present invention comprise as a binding specificity at least onefurther antibody, including, e.g., an Fab, Fab′, F(ab′)2, Fv, or a scFv.The further antibody may also be a light chain or heavy chain dimer, orany minimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al., in U.S. Pat. No. 4,946,778. The antibody mayalso be a binding-domain immunoglobulin fusion protein as disclosed inUS 2003/0118592 and US 2003/0133939.

In one embodiment, the binding specificity for an Fc receptor isprovided by a human monoclonal antibody, the binding of which is notblocked by human immunoglobulin G (IgG). As used herein, the term “lgGreceptor” refers to any of the eight γ-chain genes located onchromosome 1. These genes encode a total of twelve transmembrane orsoluble receptor isoforms which are grouped into three Fc* receptorclasses: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). In oneembodiment, the Fcγ receptor is a human high affinity FcγRI. Theproduction and characterization of these monoclonal antibodies aredescribed by Fanger et al., in WO 88/00052 and in U.S. Pat. No.4,954,617. These antibodies bind to an epitope of FcγRI, FcγRII orFcγRIII at a site which is distinct from the Fcγ binding site of thereceptor and, thus, their binding is not blocked substantially byphysiological levels of IgG. Specific anti-FcγRI antibodies useful inthe present invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. Inother embodiments, the anti-Fcγ receptor antibody is a humanized form ofmAb 22 (H22). The production and characterization of the H22 antibody isdescribed in Graziano, R. F. et al., J. Immunol. 155(10), 4996-5002(1995) and WO 94/10332. The H22 antibody producing cell line wasdeposited at the American Type Culture Collection on Nov. 4, 1992 underthe designation HA022CL1 and has the accession No. CRL 11177.

In one embodiment, the binding specificity for an Fc receptor isprovided by an antibody that binds to a human IgA receptor, e.g., an Fcαreceptor (FcαI (CD89)), the binding of which in one embodiment is notblocked by human immunoglobulin A (IgA). The term “IgA receptor” isintended to include the gene product of one α-gene (FcαRI) located onchromosome 19. This gene is known to encode several alternativelyspliced transmembrane isoforms of 55 to 110 kDa. FcαRI (CD89) isconstitutively expressed on monocytes/macrophages, eosinophilic andneutrophilic granulocytes, but not on non-effector cell populations.FcαRI has medium affinity for both IgA1 and IgA2, which is increasedupon exposure to cytokines such as G-CSF or GM-CSF (Morton, H. C. etal., Critical Reviews in Immunology 16, 423-440 (1996)). FourFcαRI-specific monoclonal antibodies, identified as A3, A59, A62 andA77, which bind FcαRI outside the IgA ligand binding domain, have beendescribed (Monteiro, R. C. et al., J. Immunol. 148, 1764 (1992)).

FcαRI, FcγRI, FcγRII and FcγRII, especially FcγRII and FcγRII, areexamples of trigger receptors for use in the present invention becausethey (1) are expressed primarily on immune effector cells, e.g.,monocytes, PMNs, macrophages and dendritic cells; (2) are expressed athigh levels (for instance 5,000-100,000 per cell); (3) are mediators ofcytotoxic activities (for instance ADCC, phagocytosis); and (4) mediateenhanced antigen presentation of antigens, including self-antigens,targeted to them.

Exemplary bispecific antibody molecules comprise (i) two antibodies onewith a specificity to CD38 and another to a second target that areconjugated together, (ii) a single antibody that has one chain specificto CD38 and a second chain specific to a second molecule, and (iii) asingle chain antibody that has specificity to CD38 and a secondmolecule. Typically, the second target/second molecule is a moleculeother than CD38. In one embodiment, the second molecule is a cancerantigen/tumor-associated antigen such as carcinoembryonic antigen (CEA),prostate specific antigen (PSA), RAGE (renal antigen), α-fetoprotein,CAMEL (CTL-recognized antigen on melanoma), CT antigens (such asMAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE,BAGE, MAGE, and SAGE), mucin antigens (e.g., MUC1, mucin-CA125, etc.),ganglioside antigens, tyrosinase, gp75, C-myc, Mart1, MelanA, MUM-1,MUM-2, MUM-3, HLA-B7, and Ep-CAM. In one embodiment, the second moleculeis a cancer-associated integrin, such as α5β3 integrin. In oneembodiment, the second molecule is an angiogenic factor or othercancer-associated growth factor, such as a vascular endothelial growthfactor (VEGF), a fibroblast growth factor (FGF), epidermal growth factor(EGF), epidermal growth factor receptor (EGFR), angiogenin, andreceptors thereof, particularly receptors associated with cancerprogression (for instance one of the HER1-HER4 receptors). Other cancerprogression-associated proteins discussed herein may also be suitablesecond molecules. In one embodiment, the second molecule is a moleculeexpressed on the surface of multiple myeloma cells such as CD138.

In one embodiment, a bispecific antibody used in the present inventionis a diabody.

Bispecific and multispecific antibodies used in the present inventionmay be made using chemical techniques (see for instance D. M. Kranz etal., PNAS USA 78, 5807 (1981)), “polydoma” techniques (See U.S. Pat. No.4,474,893) or recombinant DNA techniques.

Conjugates

In one embodiment, the present invention uses a CD38 antibody conjugatedto a therapeutic moiety, such as a cytotoxin, a chemotherapeutic drug,an immunosuppressant, or a radioisotope. Such conjugates are referred toherein as “immunoconjugates”. Immunoconjugates which include one or morecytotoxins are referred to as “immunotoxins”.

A cytotoxin or cytotoxic agent includes any agent that is detrimental to(e.g., kills) cells. For a description of these classes of drugs whichare well known in the art, and their mechanisms of action, see Goodmanet al., Goodman and Gilman's The Pharmacological Basis Of Therapeutics,8th Ed., Macmillan Publishing Co., 1990. Additional techniques relevantto the preparation of antibody immunotoxins are provided in for instanceVitetta, Immunol. Today 14, 252 (1993) and U.S. Pat. No. 5,194,594.

Suitable therapeutic agents for forming immunoconjugates useful for thepresent invention include taxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, actinomycin D, 1-dehydro-testosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin, antimetabolites (such as methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine,hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents(such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatinand other platinum derivatives, such as carboplatin), antibiotics (suchas dactinomycin (formerly actinomycin), bleomycin, daunorubicin(formerly daunomycin), doxorubicin, idarubicin, mithramycin,calicheamicin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)),diphtheria toxin and related molecules (such as diphtheria A chain andactive fragments thereof and hybrid molecules), ricin toxin (such asricin A or a deglycosylated ricin A chain toxin), cholera toxin, aShiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shigatoxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk proteaseinhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,and enomycin toxins. Therapeutic agents, which may be administered incombination with an antibody as described elsewhere herein, may also becandidates for therapeutic moieties useful for conjugation to anantibody used in the present invention. For example, the drug moiety maybe a protein or polypeptide possessing a desired biological activity.Such proteins may include, for example, an enzymatically active toxin,or active fragment thereof, such as abrin, ricin A, pseudomonasexotoxin, or diphtheria toxin; a protein such as tumor necrosis factoror interferon-γ; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6(IL-6), granulocyte macrophage colony stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), or other growth factorsand apotopic inducing protein isolated from mitochondria.

Conjugates of antibodies, and such cytotoxic moieties may be made usinga variety of bifunctional protein coupling agents. Examples of suchreagents include SPDP, IT, bifunctional derivatives of imidoesters sucha dimethyl adipimidate HCl, active esters such as disuccinimidylsuberate, aldehydes such as glutaraldehyde, bis-azido compounds such asbis (p-azidobenzoyl) hexanediamine, bis-diazonium derivatives such asbis-(p-diazoniumbenzoyl)-ethylenediamine, diisocyanates such as tolylene2,6-diisocyanate, and bis-active fluorine compounds such as1,5-difluoro-2,4-dinitrobenzene and anti-mitotic agents (e.g.,vincristine, vinblastine, docetaxel, paclitaxel and vinorelbin).

In one embodiment, the present invention uses an anti-CD38 antibody thatis conjugated to an immunomodulator, such as an immunomodulatingcytokine, stem cell growth factor, lymphotoxin (such as a TNF such asTNFα), or a hematopoietic factor. Examples of such molecules that may beuseful as conjugates include IL-1, IL-2, IL-3, IL-6, IL-10, IL-12,IL-18, and IL-21, colony stimulating factors (such as granulocyte-colonystimulating factor (G-CSF) and granulocyte macrophage-colony stimulatingfactor (GM-CSF)), interferons (such as IFNα, IFNβ, and IFNγ) the stemcell growth factor designated “S1 factor,” erythropoietin, andthrombopoietin, active fragments thereof, derivatives thereof, variantsthereof, or a combination of any thereof.

Techniques for conjugating such therapeutic moieties to antibodies, arewell known, see for instance Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al., (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985), Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), pp. 623-53(Marcel Dekker, Inc. 1987), Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al., (eds.), pp.475-506 (1985), “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.,(eds.), pp. 303-16 (Academic Press 1985) and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62, 119-58 (1982).

Additionally useful conjugate substituents include anti-cancerretinoids. Taxane conjugates (see for instance Jaime et al., AnticancerRes. 21(2A), 1119-28 (2001), cisplatin conjugates, thapsigarginconjugates, linoleic acid conjugates, calicheamicin conjugates (see forinstance Damle et al., Curr Opin Pharmacol. 3(4), 386-90 (2003),doxorubicin conjugates, geldanamycin conjugates, and the like, also maybe useful in promoting the treatment of cancer (see, generally, Trail etal., Cancer Immunol Immunother. 52(5), 328-37 (2003)).

Formulation and Mode-of-Administration

The agents used in the present invention may be formulated as apharmaceutical composition with pharmaceutically acceptable carriers ordiluents as well as any other known adjuvants and excipients inaccordance with conventional techniques such as those disclosed inRemington: The Science and Practice of Pharmacy, 19th Edition, Gennaro,Ed., Mack Publishing Co., Easton, Pa., 1995.

The pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients should be suitable for the chosencompound used in the present invention and the chosen mode ofadministration. Suitability for carriers and other components ofpharmaceutical compositions is determined based on the lack ofsignificant negative impact on the desired biological properties of thechosen compound or pharmaceutical composition (e.g., less than asubstantial impact (10% or less relative inhibition, 5% or less relativeinhibition, etc.) on antigen binding.

A pharmaceutical composition used in the present invention may alsoinclude diluents, fillers, salts, buffers, detergents (e. g., a nonionicdetergent, such as Tween-80), stabilizers, stabilizers (e. g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in apharmaceutical composition.

The actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions employed, or the ester, saltor amide thereof, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

The pharmaceutical composition may be administered by any suitable routeand mode. Suitable routes of administering a compound of the presentinvention in vivo and in vitro are well known in the art and can beselected by those of ordinary skill in the art.

The compounds used in the present invention may be administered via anysuitable route, such as an oral, nasal, inhalable, topical (includingbuccal, transdermal and sublingual), rectal, vaginal and/or parenteralroute

In one embodiment, one or more of the compounds used in the presentinvention is administered orally, for example, with an inert diluent oran assimilable edible carrier. The active ingredient may be enclosed ina hard or soft shell gelatin capsule, compressed into tablets, orincorporated directly into the subject's diet. Pharmaceuticalcompositions which are suitable for oral administration includeingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like containing such carriers asare known in the art to be appropriate. To allow oral administration, itmay be necessary to coat the compound with, or co-administer thecompound with, a material to prevent its inactivation.

In one embodiment, one or more of the compounds used in the presentinvention are administered parenterally.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and include epidermal,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.

In one embodiment, the compound is administered by intravenous orsubcutaneous injection or infusion.

In one embodiment the compounds used in the present invention areadministered in crystalline form by subcutaneous injection, cf. Yang etal., PNAS USA 100(12), 6934-6939 (2003).

Pharmaceutical compositions used in the present invention may beformulated for particular routes of administration, such as oral, nasal,topical (including buccal, transdermal and sublingual), rectal, vaginaland/or parenteral administration. The pharmaceutical compositions mayconveniently be presented in unit dosage form and may be prepared by anymethods known in the art of pharmacy. The amount of active ingredientwhich may be combined with a carrier material to produce a single dosageform will vary depending upon the subject being treated, and theparticular mode of administration. The amount of active ingredient whichmay be combined with a carrier material to produce a single dosage formwill generally be that amount of the composition which produces atherapeutic effect. Generally, out of one hundred percent, this amountwill range from about 0.01% to about 99% of active ingredient, such asfrom about 0.1% to about 70%, for instance from about 1% to about 30%.

Regardless of the route of administration selected, the compounds usedin the present invention, which may be used in the form of apharmaceutically acceptable salt or in a suitable hydrated form, and/orthe pharmaceutical compositions are formulated into pharmaceuticallyacceptable dosage forms by conventional methods known to those of skillin the art. A “pharmaceutically acceptable salt” refers to a salt thatretains the desired biological activity of the parent compound and doesnot impart any undesired toxicological effects (see for instance Berge,S. M. et al., J. Pharm. Sci. 66, 1-19 (1977)). Examples of such saltsinclude acid addition salts and base addition salts. Acid addition saltsinclude those derived from nontoxic inorganic acids, such ashydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous acids and the like, as well as from nontoxic organic acidssuch as aliphatic mono- and dicarboxylic acids, phenyl-substitutedalkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic andaromatic sulfonic acids and the like. Base addition salts include thosederived from alkaline earth metals, such as sodium, potassium,magnesium, calcium and the like, as well as from nontoxic organicamines, such as N,N′-dibenzylethylenediamine, N-methylglucamine,chloroprocaine, choline, diethanolamine, ethylenediamine, procaine andthe like.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible with a compound used inthe present invention.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions include water, saline,phosphate buffered saline, ethanol, dextrose, polyols (such as glycerol,propylene glycol, polyethylene glycol, and the like), and suitablemixtures thereof, vegetable oils, such as olive oil, corn oil, peanutoil, cottonseed oil, and sesame oil, carboxymethyl cellulose colloidalsolutions, tragacanth gum and injectable organic esters, such as ethyloleate, and/or various buffers. Other carriers are well known in thepharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions iscontemplated.

Proper fluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Pharmaceutical compositions comprises agents used in the presentinvention may also comprise pharmaceutically acceptable antioxidants forinstance (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions may also comprise isotonicity agents, suchas sugars, polyalcohols such as mannitol, sorbitol, glycerol or sodiumchloride in the compositions

Pharmaceutically acceptable diluents include saline and aqueous buffersolutions.

The pharmaceutical compositions used in the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition.Compounds of the present invention may for instance be admixed withlactose, sucrose, powders (e.g., starch powder), cellulose esters ofalkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide,sodium and calcium salts of phosphoric and sulphuric acids, acacia,gelatin, sodium alginate, polyvinylpyrrolidine, and/or polyvinylalcohol. Other examples of adjuvants are QS21, GM-CSF, SRL-172,histamine dihydrochloride, thymocartin, Tio-TEPA, monophosphoryl-lipidA/micobacteria compositions, alum, incomplete Freund's adjuvant,montanide ISA, ribi adjuvant system, TiterMax adjuvant, syntex adjuvantformulations, immune-stimulating complexes (ISCOMs), gerbu adjuvant, CpGoligodeoxynucleotides, lipopolysaccharide, andpolyinosinic:polycytidylic acid.

Prevention of presence of microorganisms may be ensured both bysterilization procedures and by the inclusion of various antibacterialand antifungal agents, for example, paraben, chlorobutanol, phenol,sorbic acid, and the like. In addition, prolonged absorption of theinjectable pharmaceutical form may be brought about by the inclusion ofagents which delay absorption such as aluminum monostearate and gelatin.

Pharmaceutical compositions comprising a compound of the presentinvention may also include a suitable salt therefore. Any suitable salt,such as an alkaline earth metal salt in any suitable form (e.g., abuffer salt), may be used in the stabilization of the compound used inthe present invention. Suitable salts typically include sodium chloride,sodium succinate, sodium sulfate, potassium chloride, magnesiumchloride, magnesium sulfate, and calcium chloride. In one embodiment, analuminum salt is used to stabilize a compound used in the presentinvention in a pharmaceutical composition, which aluminum salt also mayserve as an adjuvant when such a composition is administered to apatient.

The compounds used in the present invention may be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Such carriers may includegelatin, glyceryl monostearate, glyceryl distearate, biodegradable,biocompatible polymers such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid aloneor with a wax, or other materials well known in the art. Methods for thepreparation of such formulations are generally known to those skilled inthe art. See e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

To administer compositions by certain routes of administration, it maybe necessary to coat the compound with, or co-administer the compoundwith, a material to prevent its inactivation. For example, the compoundused in the method of the invention may be administered to a subject inan appropriate carrier, for example, liposomes, or a diluent. Liposomesinclude water-in-oil-in-water CGF emulsions as well as conventionalliposomes (Strejan et al., J. Neuroimmunol. 7, 27 (1984)).

Depending on the route of administration, the active compound may becoated in a material to protect the compound from the action of acidsand other natural conditions that may inactivate the compound. Forexample, the compound may be administered to a subject in an appropriatecarrier, for example, liposomes. Liposomes include water-in-oil-in-waterCGF emulsions as well as conventional liposomes (Strejan et al., J.Neuroimmunol. 7, 27 (1984)).

In one embodiment of the present invention, the compounds of the presentinvention are formulated in liposomes. In a further embodiment, theliposomes include a targeting moiety. In a further embodiment, thecompounds in the liposomes are delivered by bolus injection to a siteproximal to the desired area, e.g., the site of inflammation orinfection, or the site of a tumor. The composition must be fluid to theextent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi.

In one embodiment, the compounds used in the present invention may beformulated to prevent or reduce their transport across the placenta.This may be done by methods known in the art, e.g., by PEGylation of thecompounds or by use of F(ab′)₂ fragments. Further references can be madeto Cunningham-Rundles C et al., J Immunol Methods. 152, 177-190 (1992)and to Landor M., Ann Allergy Asthma Immunol 74, 279-283 (1995).

Pharmaceutically acceptable carriers for parenteral administrationinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the active compound, use thereof in thepharmaceutical compositions is contemplated. Supplementary activecompounds may also be incorporated into the compositions.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionmay be formulated as a solution, microemulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe a aqueous or nonaqueous solvent or dispersion medium containing forinstance water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. The proper fluidity may be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions may be brought about by including in thecomposition an agent that delays absorption, for example, monostearatesalts and gelatin. Sterile injectable solutions may be prepared byincorporating the active compound in the required amount in anappropriate solvent with one or a combination of ingredients e.g. asenumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients e.g. from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions may be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

In one embodiment of the methods of the invention, said at least onenon-corticosteroid chemotherapeutic agent comprises melphalan, whereinmelphalan is administered intravenously or perorally.

In another embodiment of the methods of the invention, said at least onenon-corticosteroid chemotherapeutic agent comprises a glutamic acidderivative, such as thalidomide (Thalomid®) or a thalidomide analog,e.g. CC-5013 (lenalidomide, Revlimid™) or CC4047 (Actimid™), whereinsaid glutamic acid derivative is administered perorally.

In another embodiment of the methods of the invention, said at least onenon-corticosteroid chemotherapeutic agent comprises a proteasomeinhibitor, such as bortezomib (Velcade®), wherein bortezomib isadministered intravenously.

In another embodiment of the methods of the invention, said at least onenon-corticosteroid chemotherapeutic agent comprises a vinca alkaloid,such as vincristine, wherein vincristine is administered intravenously.

In another embodiment of the methods of the invention, said at least onenon-corticosteroid chemotherapeutic agent comprises an anthracycline,such as doxorubicin, wherein doxorubicin is administered intravenously

In another embodiment of the methods of the invention, said at least onecorticosteroid comprises prednisone, wherein said prednisone isadministered perorally.

In another embodiment of the methods of the invention, said at least onecorticosteroid comprises prednisone, wherein said prednisone isadministered perorally.

Patients and Diseases to be Treated

Individuals that may be treated with the combination therapy of theinvention may for instance include human patients having disorders thatmay be corrected or ameliorated by inhibiting CD38 function, such asenzymatic activity, signal transduction, induction of cytokineexpression, induction of proliferation or differentiation, and/orinduction of lysis and/or eliminating/reducing the number of CD38expressing cells.

For example, the anti-CD38 antibodies may be used to elicit in vivo orin vitro one or more of the following biological activities: inhibitionCD38 function (such as enzymatic activity, signal transduction,induction of cytokine expression, induction of proliferation ordifferentiation, and/or induction of lysis), killing a cell expressingCD38, mediating phagocytosis or ADCC of a cell expressing CD38 in thepresence of human effector cells, and by mediating CDC of a cellexpressing CD38 in the presence of complement. or by killing CD38expressing cells by apoptosis.

In one embodiment, immunoconjugates described herein may be used totarget compounds (e.g., therapeutic agents, labels, cytotoxins,immunosuppressants, etc.) to cells which have CD38 bound to theirsurface by using such target compounds as the therapeutic moieties inimmunoconjugates of the present invention.

In one embodiment, the present invention provides methods for killingcells which have CD38 bound to their surface by administeringimmunoconjugates of the present invention.

The present invention provides methods for treating a disorder involvingcells expressing CD38 in a subject, which method comprisesadministration of a therapeutically effective amount of

i) a non-agonistic antibody which binds to CD38,

ii) at least one corticosteroid, and

iii) at least one non-corticosteroid chemotherapeutic agent,

to a subject in need thereof. Anti-CD38 antibodies are used to inhibitCD38 induced activities associated with certain disorders or toeliminate or reduce the number of cells expressing CD38.

In one embodiment of the present invention, the disorder involving cellsexpressing CD38 is a tumorigenic disorder, such as a disordercharacterized by the presence of tumor cells expressing CD38 including,for example, B cell lymphoma, plasma cell malignancies, T/NK celllymphoma and myeloid malignancies.

Examples of such tumorigenic diseases include B cell lymphoma/leukemiasincluding precursor B cell lymphoblastic leukemia/lymphoma and B cellnon-Hodgkin's lymphomas; acute promyelocytic leukemia acutelymphoblastic leukemia and mature B cell neoplasms, such as B cellchronic lymhocytic leukemia(CLL)/small lymphocytic lymphoma (SLL), Bcell acute lymphocytic leukemia, B cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicularlymphoma (FL), including low-grade, intermediate-grade and high-gradeFL, cutaneous follicle center lymphoma, marginal zone B cell lymphoma(MALT type, nodal and splenic type), hairy cell leukemia, diffuse largeB cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma,plasma cell leukemia, post-transplant lymphoproliferative disorder,Waldenström's macroglobulinemia, plasma cell leukemias and anaplasticlarge-cell lymphoma (ALCL).

In one embodiment, the disorder involving cells expressing CD38 ismultiple myeloma.

Examples of B cell non-Hodgkin's lymphomas are lymphomatoidgranulomatosis, primary effusion lymphoma, intravascular large B celllymphoma, mediastinal large B cell lymphoma, heavy chain diseases(including γ, p, and a disease), lymphomas induced by therapy withimmunosuppressive agents, such as cyclosporine-induced lymphoma, andmethotrexate-induced lymphoma.

In one embodiment of the present invention, the disorder involving cellsexpressing CD38 may be Hodgkin's lymphoma.

Examples of a disorder involving cells expressing CD38 may be amalignancy derived from T and NK cells including: mature T cell and NKcell neoplasms including T cell prolymphocytic leukemia, T cell largegranular lymphocytic leukemia, aggressive NK cell leukemia, adult T cellleukemia/lymphoma, extranodal NK/T cell lymphoma, nasal type,enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma,subcutaneous panniculitis-like T cell lymphoma, blastic NK celllymphoma, Mycosis Fungoides/Sézary Syndrome, primary cutaneous CD30positive T cell lymphoproliferative disorders (primary cutaneousanaplastic large cell lymphoma C-ALCL, lymphomatoid papulosis,borderline lesions), angioimmunoblastic T cell lymphoma, peripheral Tcell lymphoma unspecified, and anaplastic large cell lymphoma.

Examples of malignancies derived from myeloid cells include acutemyeloid leukemia, including acute promyelocytic leukemia, and chronicmyeloproliferative diseases, including chronic myeloid leukemia.

Dosages and Treatment Regimens

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result. Atherapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects. In the context ofthe present combination therapy, a therapeutic amount includes amountsthat are therapeutically effective only in combination with the othercompounds, e.g. amounts that would be too low to be effective inmonotherapy.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors. Alternatively, thisproperty of a composition may be evaluated by examining the ability ofthe compound to inhibit cell growth or to induce apoptosis by in vitroassays known to the skilled practitioner. A therapeutically effectiveamount of a therapeutic compound may decrease tumor size, or otherwiseameliorate symptoms in a subject. One of ordinary skill in the art wouldbe able to determine such amounts based on such factors as the subject'ssize, the severity of the subject's symptoms, and the particularcomposition or route of administration selected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe present invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

The efficient dosages and the dosage regimens for the anti-CD38antibodies used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art. An exemplary, non-limiting range for a therapeuticallyeffective amount of an anti-CD38 antibody used in the present inventionis about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, aboutsuch as 0.3, about 1, or about 3 mg/kg. In another embodiment, heantibody is administered in a dose of 1 mg/kg or more, such as a dose offrom 1 to 20 mg/kg, e.g. a dose of from 5 to 20 mg/kg, e.g. a dose of 8mg/kg.

A physician or veterinarian having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the medicament employed in the pharmaceutical compositionat levels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved. In general, a suitable daily dose of a compositionof the present invention will be that amount of the compound which isthe lowest dose effective to produce a therapeutic effect. Such aneffective dose will generally depend upon the factors described above.Administration may be intravenous, intramuscular, intraperitoneal, orsubcutaneous, and for instance administered proximal to the site of thetarget. If desired, the effective daily dose of a pharmaceuticalcomposition may be administered as two, three, four, five, six or moresub-doses administered separately at appropriate intervals throughoutthe day, optionally, in unit dosage forms. While it is possible for acompound of the present invention to be administered alone, it ispreferable to administer the compound as a pharmaceutical composition asdescribed above.

In one embodiment, the anti-CD38 antibody is administered by infusion ina weekly dosage of from 10 to 500 mg/m², such as of from 200 to 400mg/m². Such administration may be repeated, e.g., 1 to 8 times, such as3 to 5 times. The administration may be performed by continuous infusionover a period of from 2 to 24 hours, such as of from 2 to 12 hours.

In one embodiment, the anti-CD38 antibody is administered by slowcontinuous infusion over a long period, such as more than 24 hours, inorder to reduce toxic side effects.

In one embodiment the anti-CD38 antibody is administered in a weeklydosage of from 250 mg to 2000 mg, such as for example 300 mg, 500 mg,700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4to 6 times. The administration may be performed by continuous infusionover a period of from 2 to 24 hours, such as of from 2 to 12 hours. Suchregimen may be repeated one or more times as necessary, for example,after 6 months or 12 months. The dosage may be determined or adjusted bymeasuring the amount of compound of the present invention in the bloodupon administration by for instance taking out a biological sample andusing anti-idiotypic antibodies which target the antigen binding regionof the anti-CD38 antibody.

In a further embodiment, the anti-CD38 antibody is administered onceweekly for 2 to 12 weeks, such as for 3 to 10 weeks, such as for 4 to 8weeks.

In one embodiment, the anti-CD38 antibody is administered by maintenancetherapy, such as, e.g., once a week for a period of 6 months or more.

In one embodiment, the anti-CD38 antibody is administered by a regimenincluding one infusion of an anti-CD38 antibody followed by an infusionof an anti-CD38 antibody conjugated to a radioisotope. The regimen maybe repeated, e.g., 7 to 9 days later.

As non-limiting examples, treatment according to the present inventionmay be provided as a daily dosage of an antibody in an amount of about0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on atleast one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 afterinitiation of treatment, or any combination thereof, using single ordivided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combinationthereof.

In one embodiment of the methods of the invention, said at least onenon-corticosteroid chemotherapeutic agent comprises melphalan, and saidat least one corticosteroid comprises prednisone. Typically, melphalanis dosed intravenously (IV), but it can be used perorally (PO), e.g. inthe range of 0.2-0.25 mg/kg per day or e.g. 7-9 mg/m2). Prednisone maye.g. be dosed at 2 mg/kg for 4 days every 4-6 weeks (Alexanian et al., JAm Med Assoc 1969; 208:1680). In other embodiments, melphalan can beused in high dose regimen in single doses up to 140 mg/m2 (IV) orintermediate doses in range of 25 to 75 mg/m2 (IV), one example is 40mg/d administered days 1-4, 9-12 and 17-20 every 5 week cycle(Tsakanikas et al., Oncology 1991; 48:369, Richardson P G Am J Oncol2005; 4:737).

In another embodiment of the methods of the invention, said at least onenon-corticosteroid chemotherapeutic agent comprises thalidomide(Thalomid®), and said at least one corticosteroid comprisesdexamethasone. Thalidomide can e.g. be used a in dose of 200 mg/d (PO),or e.g. in a range from 50 to 400 mg/d together with e.g. a dose ofdexamethasone of 40 mg/d either administered daily or administeredsequentially, e.g. day 1-4, 9-12 and 17-20 of each 28-day cycle.(Rajkumar S V J Clin Oncol 2006; 24:431).

In another embodiment of the methods of the invention, said at least onenon-corticosteroid chemotherapeutic agent comprises lenalidomide, andsaid at least one corticosteroid comprises dexamethasone. Lenalidomidecan e.g. be administered in doses of 25 mg/d administered daily (PO) anddexamethasone e.g. in a range of 40 mg/d administered (PO) e.g. on day1-4, 9-12 and 17-20 of 28-day cycle optionally later only on day 1-4 ofeach cycle (Rajkumar S V, ASH 2004).

In another embodiment of the methods of the invention, said at least onenon-corticosteroid chemotherapeutic agent comprises bortezomib(Velcade®). Bortezomib can e.g. be used combination with dexamethasone.This combination can be used both in induction and maintenance setting.One example is bortezomib 1.3 mg/m2 on days 1, 4, 8 and 11 every 21 daycycle (induction phase, normally up to 8 cycles) followed by days 1, 8,11, 15 and 22 every 5 week cycle for maintenance (Richardson P G N EnglJ Med 2005; 352:2487).

In another embodiment of the methods of the invention, said at least onenon-corticosteroid chemotherapeutic agent comprises vincristine anddoxorubicin, and said at least one corticosteroid comprisesdexamethasone. Vincristine may e.g. be administered by continuous IVinfusion, 0.4 mg per day (days 1-4 on every 4 week cycle) anddoxorubicin e.g. in a dose of 9 mg/m2/d continuous IV infusions on days1-4 in every 4 week cycle. Dexametasone can e.g. be dosed 40 mg on days1-4, 9-12 and 17-21 every 4 week cycle. Alternatively pegylatedliposomal doxorubicin (e.g. in a dose of 40 mg/m2 on day 1 in a weekcycle) can be used (Rifkin Cancer 2006; 106:848).

Further Combinations

The combination therapy of the invention may be further combined withother medicaments, i.e., combined with further therapeutic agentsrelevant for the disease or condition to be treated. Such administrationmay be simultaneous, separate or sequential. For simultaneousadministration the agents may be administered as one compositions or asseparate compositions, as appropriate.

Accordingly, the present invention provides methods for treating adisorder involving cells expressing CD38 as described above, whichmethods comprise the triple therapy of the present invention combinedwith one or more additional therapeutic agents as described below.

In one embodiment, the combination therapy of the invention furtherincludes administration of at least one chemotherapeutic agent, at leastone anti-inflammatory agent, or at least one immunosuppressive and/orimmunomodulatory agent.

In one embodiment, such a chemotherapeutic agent may be selected from anantimetabolite, such as methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea,asparaginase, gemcitabine, cladribine and similar agents.

In one embodiment, such a chemotherapeutic agent may be selected from anantibiotic, such as dactinomycin (formerly actinomycin), bleomycin,daunorubicin (formerly daunomycin), idarubicin, mithramycin, mitomycin,mitoxantrone, plicamycin, anthramycin (AMC) and similar agents.

In one embodiment, such a chemotherapeutic agent may be selected from ananti-mitotic agent, such as taxanes, for instance docetaxel, andpaclitaxel.

In one embodiment, such a chemotherapeutic agent may be selected from atopoisomerase inhibitor, such as topotecan.

In one embodiment, such a chemotherapeutic agent may be selected from agrowth factor inhibitor, such as an inhibitor of ErbB1 (EGFR) (such asgefitinib (Iressa®), cetuximab (Erbitux®), erlotinib (Tarceva®), 2F8(disclosed in WO 2002/100348) and similar agents), an inhibitor of ErbB2(Her2/neu) (such as trastuzumab (Herceptin®) and similar agents) andsimilar agents. In one embodiment, such a growth factor inhibitor may bea farnesyl transferase inhibitor, such as SCH-66336 and R115777. In oneembodiment, such a growth factor inhibitor may be a vascular endothelialgrowth factor (VEGF) inhibitor, such as bevacizumab (Avastin®).

In one embodiment, such a chemotherapeutic agent may be a tyrosinekinase inhibitor, such as imatinib (Glivec, Gleevec ST1571), lapatinib,PTK787/ZK222584 and similar agents.

In one embodiment, such a chemotherapeutic agent may be a histonedeacetylase inhibitor. Examples of such histone deacetylase inhibitorsinclude hydroxamic acid-based hybrid polar compounds, such as SAHA(suberoylanilide hydroxamic acid).

In one embodiment, such a chemotherapeutic agent may be a P38a MAPkinase inhibitor, such as SCIO-469.

In a further embodiment, the combination therapy of the inventionfurther includes administration of at least one inhibitor ofangiogenesis, neovascularization, and/or other vascularization to asubject in need thereof

Examples of such angiogenesis inhibitors are urokinase inhibitors,matrix metalloprotease inhibitors (such as marimastat, neovastat, BAY12-9566, AG 3340, BMS-275291 and similar agents), inhibitors ofendothelial cell migration and proliferation (such as TNP-470,squalamine, 2-methoxyestradiol, combretastatins, endostatin,angiostatin, penicillamine, SCH66336 (Schering-Plough Corp, Madison,N.J.), R115777 (Janssen Pharmaceutica, Inc, Titusville, N.J.) andsimilar agents), antagonists of angiogenic growth factors (such as suchas ZD6474, SU6668, antibodies against angiogenic agents and/or theirreceptors (such as VEGF, bFGF, and angiopoietin-1), Sugen 5416, SU5402,antiangiogenic ribozyme (such as angiozyme), interferon α (such asinterferon α2a), suramin and similar agents), VEGF-R kinase inhibitorsand other anti-angiogenic tyrosine kinase inhibitors (such as SU011248),inhibitors of endothelial-specific integrin/survival signaling (such asvitaxin and similar agents), copper antagonists/chelators (such astetrathiomolybdate, captopril and similar agents), carboxyamido-triazole(CAI), ABT-627, CM101, interleukin-12 (IL-12), IM862, PNU145156E as wellas nucleotide molecules inhibiting angiogenesis (such asantisense-VEGF-cDNA, cDNA coding for angiostatin, cDNA coding for p53and cDNA coding for deficient VEGF receptor-2) and similar agents.

Other examples of such inhibitors of angiogenesis, neovascularization,and/or other vascularization are anti-angiogenic heparin derivatives andrelated molecules (e.g., heperinase Ill), temozolomide, NK4, macrophagemigration inhibitory factor (MIF), cyclooxygenase-2 inhibitors,inhibitors of hypoxia-inducible factor 1, anti-angiogenic soyisoflavones, oltipraz, fumagillin and analogs thereof, somatostatinanalogues, pentosan polysulfate, tecogalan sodium, dalteparin,tumstatin, thrombospondin, NM-3, combrestatin, canstatin, avastatin,antibodies against other relevant targets (such as anti-alpha-v/beta-3integrin and anti-kininostatin mAbs) and similar agents.

In a further embodiment, the combination therapy of the inventionfurther includes administration of an anti-cancer immunogen, such as acancer antigen/tumor-associated antigen (e.g., epithelial cell adhesionmolecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen(CEA), tumor-associated glycoprotein 72 (TAG-72), gp100, Melan-A,MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines (e.g., humanpapillomavirus vaccines), tumor-derived heat shock proteins, and similaragents. A number of other suitable cancer antigens/tumor-associatedantigens described elsewhere herein and similar molecules known in theart may also or alternatively be used in such embodiment. Anti-cancerimmunogenic peptides also include anti-idiotypic “vaccines” such as BEC2anti-idiotypic antibodies, Mitumomab, CeaVac and related anti-idiotypicantibodies, anti-idiotypic antibody to MG7 antibody, and otheranti-cancer anti-idiotypic antibodies (see for instance Birebent et al.,Vaccine. 21(15), 1601-12 (2003), Li et al., Chin Med J (Engl). 114(9),962-6 (2001), Schmitt et al., Hybridoma. 13(5), 389-96 (1994), Maloneyet al., Hybridoma. 4(3), 191-209 (1985), Raychardhuri et al., J Immunol.137(5), 1743-9 (1986), Pohl et al., Int J Cancer. 50(6), 958-67 (1992),Bohlen et al., Cytokines Mol Ther. 2(4), 231-8 (1996) and Maruyama, JImmunol Methods. 264(1-2), 121-33 (2002)). Such anti-idiotypic Abs mayoptionally be conjugated to a carrier, which may be a synthetic(typically inert) molecule carrier, a protein (for instance keyholelimpet hemocyanin (KLH) (see for instance Ochi et al., Eur J Immunol.17(11), 1645-8 (1987)), or a cell (for instance a red blood cell—see forinstance Wi et al., J Immunol Methods. 122(2), 227-34 (1989)).

In a further embodiment, the combination therapy of the inventionfurther includes administration of a bisphosphonate. Examples ofpotentially suitable biphosphonates are pamidronate (Aredia®),zoledronic acid (Zometa®), clodronate (Bonefos®), risendronate(Actonel®), ibandronate (Boniva®), etidronate (Didronel®), alendronate(Fosamax®), tiludronate (Skelid®), incadronate (YamanouchiPharmaceutical) and minodronate (YM529, Yamanouchi).

In a further embodiment, the combination therapy of the inventionfurther includes administration of a colony stimulating factor. Examplesof suitable colony stimulating factors are granulocyte-colonystimulating factors (G-CSF), such as filgrastim (Neupogen®) andpegfilgrastim (Neulasta®), and granulocyte macrophage-colony stimulatingfactors (GM-CSF) such as sargramostim (Leukine®).

In a further embodiment, the combination therapy of the inventionfurther includes administration of an erythropoietic agent. Examples ofsuitable erythropoietic agents are erythropoietin (EPO), such as epoetinalfa (for instance Procrit®, Epogen®, and Eprex®) and epoetin beta (forinstance NeoRecormon®) and erythropoiesis-stimulating proteins (forinstance Aranesp®).

In a further embodiment, the combination therapy of the inventionfurther includes administration of an anti-cancer cytokine, chemokine,or combination thereof. Examples of suitable cytokines and growthfactors include IFNγ, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13,IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα(e.g., INFα2b), IFNβ, GM-CSF, CD40L, Flt3 ligand, stem cell factor,ancestim, and TNFα. Suitable chemokines may include Glu-Leu-Arg(ELR)-negative chemokines such as IP-10, MCP-3, MIG, and SDF-1α from thehuman CXC and C-C chemokine families. Suitable cytokines includecytokine derivatives, cytokine variants, cytokine fragments, andcytokine fusion proteins.

In a further embodiment, the combination therapy of the inventionfurther includes administration of an agent that modulates, e.g.,enhances or inhibits, the expression or activity of Fcα or Fcγreceptors. Examples of agents suitable for this use includeinterleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),granulocyte colony-stimulating factor (G-CSF), such as filgrastim(Neupogen®) and pegfilgrastim (Neulasta®), and granulocytemacrophage-colony stimulating factors (GM-CSF) such as sargramostim(Leukine®), interferon-γ (IFN-γ), and tumor necrosis factor (TNF).

In a further embodiment, the combination therapy of the inventionfurther includes administration of a cell cycle control/apoptosisregulator (or “regulating agent”). A cell cycle control/apoptosisregulator may include molecules (i) that target and modulate cell cyclecontrol/apoptosis regulators such as cdc-25 (such as NSC 663284), (ii)cyclin-dependent kinases that overstimulate the cell cycle (such asflavopiridol (L868275, HMR1275), 7-hydroxystaurosporine (UCN-01,KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerasemodulators (such as BIBR1532, SOT-095, GRN163 and compositions describedin for instance U.S. Pat. Nos. 6,440,735 and 6,713,055). Non-limitingexamples of molecules that interfere with apoptotic pathways includeTNF-related apoptosis-inducing ligand (TRAIL)/apoptosis-2 ligand(Apo-2L), agents inducing NF-κB blockade leading to inhibition of IL-6production, antibodies that activate TRAIL receptors, IFNs, □ anti-senseBcl-2, and As₂O₃(arsenic trioxide, Trisenox®).

In a further embodiment, the combination therapy of the inventionfurther includes administration of a hormonal regulating agent, such asagents useful for anti-androgen and anti-estrogen therapy. Examples ofsuch hormonal regulating agents are tamoxifen, idoxifene, fulvestrant,droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinylestradiol/estinyl, an antiandrogene (such as flutaminde/eulexin), aprogestin (such as such as hydroxyprogesterone caproate,medroxyprogesterone/provera, megestrol acepate/megace), anadrenocorticosteroid (such as hydrocortisone, prednisone), luteinizinghormone-releasing hormone (and analogs thereof and other LHRH agonistssuch as buserelin and goserelin), an aromatase inhibitor (such asanastrazole/arimidex, aminoglutethimide/cytraden, exemestane), a hormoneinhibitor (such as octreotide/-sandostatin) and similar agents.

In a further embodiment, the combination therapy of the inventionfurther includes administration of an anti-anergic agent (for instancesmall molecule compounds, proteins, glycoproteins, or antibodies thatbreak tolerance to tumor and cancer antigens). Examples of suchcompounds are molecules that block the activity of CTLA-4, such asMDX-010 (Phan et al., PNAS USA 100, 8372 (2003)).

In a further embodiment, the combination therapy of the inventionfurther includes administration of a tumor suppressor gene-containingnucleic acid or vector such as a replication-deficient adenovirusencoding human recombinant wild-type p53/SCH58500, etc.; antisensenucleic acids targeted to oncogenes, mutated, or deregulated genes; orsiRNA targeted to mutated or deregulated genes. Examples of tumorsuppressor targets include, for example, BRCA1, RB1, BRCA2, DPC4(Smad4), MSH2, MLH1, and DCC.

In a further embodiment, the combination therapy of the inventionfurther includes administration of an anti-cancer nucleic acid, such asgenasense (augmerosen/G3139), LY900003 (ISIS 3521), ISIS 2503, OGX-011(ISIS 112989), LE-AON/LEraf-AON (liposome encapsulated c-raf antisenseoligonucleotide/ISIS-5132), MG98, and other antisense nucleic acids thattarget PKCα, clusterin, IGFBPs, protein kinase A, cyclin D1, or Bcl-2h.

In a further embodiment, the combination therapy of the inventionfurther includes administration of an anti-cancer inhibitory RNAmolecule (see for instance Lin et al., Curr Cancer Drug Targets. 1(3),241-7 (2001), Erratum in: Curr Cancer Drug Targets. 3(3), 237 (2003),Lima et al., Cancer Gene Ther. 11(5), 309-16 (2004), Grzmil et al., IntJ Oncol. 4(1), 97-105 (2004), Collis et al., Int J Radiat Oncol BiolPhys. 57(2 Suppl), S144 (2003), Yang et al., Oncogene. 22(36), 5694-701(2003) and Zhang et al., Biochem Biophys Res Commun. 303(4), 1169-78(2003)).

In a further embodiment, the combination therapy of the inventionfurther includes administration of a virus, viral proteins, and thelike. Replication-deficient viruses, that generally are capable of oneor only a few rounds of replication in vivo, and that are targeted totumor cells, may for instance be useful components of such compositionsand methods. Such viral agents may comprise or be associated withnucleic acids encoding immunostimulants, such as GM-CSF and/or IL-2.Both naturally oncolytic and such recombinant oncolytic viruses (forinstance HSV-1 viruses, reoviruses, replication-deficient andreplication-sensitive adenovirus, etc.) may be useful components of suchmethods and compositions (see for instance Shah et al., J Neurooncol.65(3), 203-26 (2003), Stiles et al., Surgery. 134(2), 357-64 (2003),Sunarmura et al., Pancreas. 28(3), 326-9 (2004), Teshigahara et al., JSurg Oncol. 85(1), 42-7 (2004), Varghese et al., Cancer Gene Ther.9(12), 967-78 (2002), Wildner et al., Cancer Res. 59(2), 410-3 (1999),Yamanaka, Int J Oncol. 24(4), 919-23 (2004) and Zwiebel et al., SeminOncol. 28(4), 336-43 (2001).

In a further embodiment, the combination therapy of the invention mayfurther involve “whole cell and “adoptive” immunotherapy methods. Forinstance, such methods may comprise infusion or re-infusion of immunesystem cells (for instance tumor-infiltrating lymphocytes (TILs), suchas CD4⁺ and/or CD8⁺ T cells (for instance T cells expanded withtumor-specific antigens and/or genetic enhancements),antibody-expressing B cells or other antibody producing/presentingcells, dendritic cells (e.g., anti-cytokine expressing recombinantdendritic cells, dendritic cells cultured with a DC-expanding agent suchas GM-CSF and/or Flt3-L, and/or tumor-associated antigen-loadeddendritic cells), anti-tumor NK cells, so-called hybrid cells, orcombinations thereof. Cell lysates may also be useful in such methodsand compositions. Cellular “vaccines” in clinical trials that may beuseful in such aspects include Canvaxin™, APC-8015 (Dendreon), HSPPC-96(Antigenics), and Melacine® cell lysates. Antigens shed from cancercells, and mixtures thereof (see for instance Bystryn et al., ClinicalCancer Research Vol. 7, 1882-1887, July 2001), optionally admixed withadjuvants such as alum, may also be components in such methods andcombination compositions.

In a further embodiment, the combination therapy of the inventionfurther includes the application of an internal vaccination method.Internal vaccination refers to induced tumor or cancer cell death, suchas drug-induced or radiation-induced cell death of tumor cells, in apatient, that typically leads to elicitation of an immune responsedirected towards (i) the tumor cells as a whole or (ii) parts of thetumor cells including (a) secreted proteins, glycoproteins or otherproducts, (b) membrane-associated proteins or glycoproteins or othercomponents associated with or inserted in membranes, and/or (c)intracellular proteins or other intracellular components. An internalvaccination-induced immune response may be humoral (i.e.antibody-complement-mediated) or cell-mediated (e.g., the developmentand/or increase of endogenous cytotoxic T lymphocytes that recognize theinternally killed tumor cells or parts thereof).

In a further embodiment, the combination therapy of the inventionfurther includes administration of complement. Accordingly, the use ofcompositions comprising anti-CD38 antibodies with serum or complement isalso within the scope of the present invention. In these compositionsthe complement is located in close proximity to the anti-CD38 antibody,for instance by conjugation or may be suited for simultaneousadministration. Alternatively, the anti-CD38 antibodies and thecomplement or serum may be administered separately.

In a further embodiment, the combination therapy of the inventionfurther includes administration of differentiation inducing agents,retinoic acid and retinoic acid analogues (such as all trans retinoicacid, 13-cis retinoic acid and similar agents), vitamin D analogues(such as seocalcitol and similar agents), inhibitors of ErbB3, ErbB4,IGF-IR, insulin receptor, PDGFRa, PDGFRbeta, Flk2, Flt4, FGFR1, FGFR2,FGFR3, FGFR4, TRKA, TRKC, c-met, Ron, Sea, Tie, Tie2, Eph, Ret, Ros,Alk, LTK, PTK7 and similar agents.

In a further embodiment, the combination therapy of the inventionfurther includes administration of a cathepsin B, modulators ofcathepsin D dehydrogenase activity, glutathione-S-transferase (such asglutacylcysteine synthetase and lactate dehydrogenase), or similaragents.

In a further embodiment, the combination therapy of the inventionfurther includes administration of estramustine or epirubicin.

In a further embodiment, the combination therapy of the inventionfurther includes administration of a HSP90 inhibitor like 17-allyl aminogeld-anamycin, antibodies directed against a tumor antigen such as PSA,CA125, KSA, etc., integrins like integrin β1, inhibitors of VCAM orsimilar agents

In a further embodiment, the combination therapy of the inventionfurther includes administration of calcineurin-inhibitors (such asvalspodar, PSC 833 and other MDR-1 or p-glycoprotein inhibitors),TOR-inhibitors (such as sirolimus, everolimus and rapamycin). andinhibitors of “lymphocyte homing” mechanisms (such as FTY720), andagents with effects on cell signaling such as adhesion moleculeinhibitors (for instance anti-LFA, etc.).

In a further embodiment, the combination therapy of the inventionfurther includes radiotherapy.

Radiotherapy may comprise radiation or associated administration ofradiopharmaceuticals to a patient is provided. The source of radiationmay be either external or internal to the patient being treated(radiation treatment may, for example, be in the form of external beamradiation therapy (EBRT), brachytherapy (BT) or skeletal targetedradiotherapy). Radioactive elements that may be used in practicing suchmethods include, e.g., radium, cesium-137, iridium-192, americium-241,gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131,and indium-111.

In a further embodiment, the combination therapy of the inventionfurther includes autologous peripheral stem cell or bone marrowtransplantation.

In a further embodiment, the combination therapy of the inventionfurther includes orthopedic intervention.

Orthopedic interventions may be used in the treatment of a disorderinvolving cells expressing CD38, such as multiple myeloma, to helpcontrol pain or retain function or mobility. Such interventions mayinclude physical therapy, splinting of bones to prevent or treatfractures, or surgical procedures (minor or major) to repair fractures.

In a further embodiment, the combination therapy of the inventionfurther includes delivery of one or more agents that promote access ofthe CD38 antibody or combination composition to the interior of a tumor.Such methods may for example be performed in association with thedelivery of a relaxin, which is capable of relaxing a tumor (see forinstance U.S. Pat. No. 6,719,977). In one embodiment, the anti-CD38antibody used in the present invention may be bonded to a cellpenetrating peptide (CPP). Cell penetrating peptides and relatedpeptides (such as engineered cell penetrating antibodies) are describedin for instance Zhao et al., J Immunol Methods. 254(1-2), 137-45 (2001),Hong et al., Cancer Res. 60(23), 6551-6 (2000). Lindgren et al., BiochemJ. 377(Pt 1), 69-76 (2004), Buerger et al., J Cancer Res Clin Oncol.129(12), 669-75 (2003), Pooga et al., FASEB J. 12(1), 67-77 (1998) andTseng et al., Mol Pharmacol. 62(4), 864-72 (2002).

In a further embodiment, the combination therapy of the inventionfurther includes administration of at least one anti-inflammatory agent.

In one embodiment such an anti-inflammatory agent may be selected from asteroidal drug and a NSAID (nonsteroidal anti-inflammatory drug).

In one embodiment such an anti-inflammatory agent may be selected fromaspirin and other salicylates, Cox-2 inhibitors (such as rofecoxib andcelecoxib), NSAIDs (such as ibuprofen, fenoprofen, naproxen, sulindac,diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone, etodolac,oxaprozin, and indomethacin), anti-IL6R antibodies, anti-IL8 antibodies(e.g. 10F8 described in WO2004/058797), anti-IL15 antibodies, anti-IL15Rantibodies, anti-CD4 antibodies, anti-CD11a antibodies (e.g.,efalizumab), anti-alpha-4/beta-1 integrin (V_(L)A4) antibodies (e.gnatalizumab), CTLA4-lg for the treatment of inflammatory diseases,prednisolone, prednisone, disease modifying antirheumatic drugs (DMARDs)such as methotrexate, hydroxychloroquine, sulfasalazine, pyrimidinesynthesis inhibitors (such as leflunomide), IL-1 receptor blockingagents (such as anakinra), TNF-α blocking agents (such as etanercept,infliximab, and adalimumab) and similar agents.

In a further embodiment, the combination therapy of the inventionfurther includes administration of at least one immunosuppressive and/orimmunomodulatory agent to a subject in need thereof.

In one embodiment, such an immunosuppressive and/or immunomodulatoryagent may be selected from cyclosporine, azathioprine, mycophenolicacid, mycophenolate mofetil, corticosteroids such as prednisone,methotrexate, gold salts, sulfasalazine, antimalarials, brequinar,leflunomide, mizoribine, 15-deoxyspergualine, 6-mercaptopurine,cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocyteglobulin, thymopentin, thymosin-α and similar agents.

In one embodiment, such an immunosuppressive and/or immunomodulatoryagent may be selected from immunosuppressive antibodies, such asantibodies binding to p75 of the IL-2 receptor, or antibodies binding tofor instance MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFNγ, TNF-α,IL-4, IL-5, IL-6R, IL-6; IGF, IGFR1, IL-7, IL-8, IL-10, CD11a, or CD58,or antibodies binding to their ligands.

In one embodiment, such an immunosuppressive and/or immunomodulatoryagent may be selected from soluble IL-15R, IL-10, B7 molecules (B7-1,B7-2, variants thereof, and fragments thereof), ICOS, and OX40, aninhibitor of a negative T cell regulator (such as an antibody againstCTLA4) and similar agents.

In a further embodiment, the combination therapy of the inventionfurther includes administration of an anti-C3b(i) antibody.

In a further embodiment, the combination therapy of the inventionfurther includes administration of histone deacetylase inhibitors (forinstance phenylbutyrate) and/or DNA repair agents (for instance DNArepair enzymes and related compositions such as dimericine).

In a further embodiment, the combination therapy of the inventionfurther includes anti-cancer directed photodynamic therapy (for instanceanti-cancer laser therapy -which optionally may be practiced with theuse of photosensitizing agent, see, for instance Zhang et al., J ControlRelease. 93(2), 141-50 (2003)), anti-cancer sound-wave and shock-wavetherapies (see for instance Kambe et al., Hum Cell. 10(1), 87-94(1997)), and/or anti-cancer nutraceutical therapy (see for instanceRoudebush et al., Vet Clin North Am Small Anim Pract. 34(1), 249-69,viii (2004) and Rafi, Nutrition. 20(1), 78-82 (2004).

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

All patents, pending patent applications and other publications citedherein are hereby incorporated by reference in their entirety.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting.

EXAMPLES Example 1 Manufacturing Luciferase-Transfected (Daudi-Luc)Cells

Culture of Daudi cells (originating from Burkitt's lymphoma) wascultured in RPMI 1640 culture medium supplemented with 10% FCS (OptimumC241, Wisent Inc., St. Bruno, QC, Canada), 2 mM L-glutamine, 100 IU/mlpenicillin, 100 mg/ml streptomycin, 1 mM sodium pyruvate (all derivedfrom Gibco BRL, Life Technologies, Paisley, Scotland). Medium wasrefreshed twice a week. Before transfection, cells were split and seededout at 1-1.5×10⁶ cells/ml to ensure viability and optimal growth.

Luciferase Transfection

8.2×10⁶ CD38⁺ Daudi cells were taken up in 350 μl RPMI (supplementedwith 10% dFCS, Gibco BRL) and transferred to an electroporation cuvet(Biorad, Hemel Hempstead, Herts, UK). Then, 40 μg gWIZ luciferase fromGTS (Aldevron, Fargo, N. Dak., USA) and 10 μg pPur vector (BDBiosciences, Alphen a/d Rijn, The Netherlands), which confers puromycinresistance, were added. After resting cells on ice for 10 minutes, cellswere electroporated (250 V, 950 μF; Gene Pulser II, Biorad LaboratoriesGmbH, Minchen, Germany). Cells were again rested on ice, and taken up in40 ml RPMI (supplemented with 10% FCS). Then, cells were plated out in96-well tissue culture plates (100 μl per well). After 48 hours,puromycin (final concentration: 1 μg/ml; Sigma-Aldrich Chemie BV,Zwijndrecht, The Netherlands) was added. Puromycin-resistant clones werefurther grown in 24-well tissue culture plates.

Determination of Luciferase Activity

Luciferase activity of cells was determined using the Luciferase AssaySystem (# E4030, Promega, Madison, Wis., USA). 1×10⁵ cells werecentrifuged (13.500 rpm, 1 min) in an eppendorf centrifuge, and thepellet was washed in 100 μl PBS. After centrifugation (13.500 rpm, 1min), pellet was lysed with 20 μl Reporter Lysis Buffer (Promega),frozen and thawed. After centrifugation (13,500 rpm, 1 min), 20 μlsupernatant was discarded, and 100 μl luciferase assay reagent was added(in special luminometer tubes, Promega). Luminescence was measured (10sec) in a luminometer (LB9507, Berthold, Vilvoorde, Belgium).

Example 2 Immunization of Mice and Generation of Hybridomas

Immunization Protocol for -003

HCo12 mice were immunized every fortnight with 20 μg purified HA-CD38.The first immunization was performed i.p. in the presence of 100 μl PBS,mixed with 100 μl Complete Freund's Adjuvant (CFA). After this firstimmunization, subsequent boosts (13×) with purified HA-CD38 wereperformed in the presence of 100 μl PBS, mixed with 100 μl IncompleteFreund's Adjuvant (IFA) alternating s.c. and i.p. After titerdevelopment, mice were boosted with 20 μg HA-CD38 in PBS, i.v.

Immunization Protocol for -005 and -024

HCo12 mice were immunized every fortnight with 20 μg purified HA-CD38alternating with NIH-3T3-CD38 transfected cells. The first immunizationwas performed with 5×10⁶ cells in 100 μl PBS, mixed with 100 μl CFA,i.p., the second and following immunizations with HA-CD38 s.c., in thepresence of 100 μl PBS, mixed with 100 μl IFA.

The following immunizations with transfected cells were performed in thepresence of 200 μl PBS. After titer development, mice were boosted with20 μg HA-CD38 in PBS, i.v.

Generation of Hybridomas Producing Human Monoclonal Antibodies to CD38

The mouse splenocytes were isolated from HCo12 mice and fused with PEGto a mouse myeloma cell line based upon standard protocols. Theresulting hybridomas were then screened for human antibody production byELISA and for CD38 specificity using human CD38-transfected NS/0 cellsby FACS analysis and recombinant HA-CD38 protein binding by ELISA. Threehybridoma cell lines were selected expressing the human monoclonalanti-CD38 antibodies, -003, -005 and -024, respectively.

Example 3

Transfection of NIH cells with CD38

The vector (pclpuroCD38) for producing NIH-3T3-CD38 cells was obtainedfrom Prof. M. Glennie (Tenovus Research Laboratory, Southampton GeneralHospital, Southampton, UK). NIH-3T3 cells (DSMZ, ACC 59; 150,000cells/well; 0.5 ml; 96-well flat-bottom plates, Greiner) were culturedin DMEM (supplemented with glucose [4.5 g/l], 10% FCS, L-glutamine,Na-pyruvate; BioWhittaker) for 24 h. Then, DNA (0.8 μg) andlipofectamine (Invitrogen, Breda, The Netherlands) were diluted in DMEM,and mixed (20 min, RT). Thereafter, the mixture (100 μl) was added toeach well and incubated (ON, 37° C.).

Screening for CD38 Expression

NIH-3T3-CD38 cells were washed (in 1 ml PBS) and trypsinized (200 μl,trypsin-EDTA, BioWhittaker). Then, 1 ml of DMEM was added and themixture pipetted into FACS tubes. After centrifugation (1200 rpm, 5min), cells were washed in FACS Buffer (FB; PBS, 0.05% BSA, 0.02% NaN₃)and resuspended in 1 ml FB. After centrifugation (1200 rpm, 5 min),supernatant was removed and mouse anti-human CD38-PE was added (1/50dilution, Sanquin, Amsterdam, The Netherlands). After washing the cellstwice in FB, cells were resuspended in FB for acquisition by flowcytometry.

Expansion and Selection

After trypsine treatment, cells were transferred to T25 flasks (Greiner)in DMEM (supplemented with glucose 4.5 g/l, 2 mM L-glutamine, andpuromycin (2 μg/ml) BioWhittaker). Puromycin-resistant cells were testedfor stable CD38 expression by flow cytometry after two weeks onpuromycin-containing medium. NIH-3T3-CD38 selected cells were subclonedby limiting dilution. After expanding these cells, all 15 NIH-3T3-CD38clones were screened for CD38 expression. CD38 high NIH-3T3-CD38 cellswere frozen in liquid nitrogen (−80° C.) until use.

Culture of NIH-3T3-CD38 Cells

Cells are cultured in DMEM (supplemented with glucose (4.5 g/l), 10%FCS, 2 mM L-glutamine, Na-pyruvate, penicillin, streptomycin). Cells arepassaged twice a week by use of trypsin/EDTA and seeded in aconcentration of 1×10⁶ cells/T75 flask. CD38high NIH-3T3-CD38 cells werefrozen in liquid nitrogen (−80° C.) until use.

Purification of HA-CD38 Antigen

Sepharose 4B (Amersham Bioscience, Uppsala, Sweden) was coupled withanti-CD38 antibody (Serotec, Oxford, UK). Column (column tube HR5/20 waspacked to 12 cm bedheight, column volume 2.4 ml; maximum flow rate 0.5ml/min) was equilibrated with at least 5 column volumes (CV) of PBS.Sample was filtrated and loaded to the column. Column was washed withPBS until signal returned to baseline (approximately 3 CV). Elution wascarried out with 0.1M glycine at pH 2. Eluted fractions were neutralizedwith 1% (v/v) 2 M Tris-HCl, pH 9.

Purification of anti-CD38 Antibodies

Human anti-CD38 antibodies were purified from tissue culturesupernatants.

First, the supernatants were filtered over 0.20 μM dead-end filter.Then, the supernatant was loaded on a 5 ml Protein A column (rProtein AFF, Amersham Bioscience) and eluted with 0.1 M citric acid-NaOH, pH 3.The eluate was immediately neutralized with 2 M Tris-HCl, pH 9 anddialyzed O/N to 12.6 mM sodium phosphate, 140 mM NaCl, pH 7.4 (B. Braun,Oss, The Netherlands). After dialysis samples were sterile filtered over0.20 μM dead-end filter.

Purification of His-CD38 Batches

The protein is present in cell culture supernatant ofHis-CD38-expressing cells, with a DNA construct containing the sequencefor the extracellular domain of CD38. An additional poly-His-tagsequence is included in the constructs and present at the N-terminus ofthe protein. This tag enables purification with immobilized metalaffinity chromatography. In this process, a chelator fixed onto thechromatographic resin is charged with Co²⁺ cations. Particularly, asequence that includes 6 histidine amino acids strongly binds Co²⁺.Therefore the His-tagged CD38 proteins bind strongly to such a column,while other proteins present in the culture supernatant will flowthrough the column or will be washed away. The strongly bound His-taggedCD38 proteins are then eluted with a buffer containing imidazole, whichcompetes with the binding of His to Co²⁺. When sufficient His-CD38 ispurified, the eluent is removed from the protein by buffer exchange on adesalting column.

Example 4 Binding of -003, -005, and -024 to CD38-Transfected CHO(CHO-CD38) Cells, to Daudi-Luc Cells and to Fresh Multiple Myeloma (MM)Tumor Cells

After harvesting and counting, Daudi-luc cells, CHO cells transfectedwith CD38 and control CHO cells were resuspended in PBS (1×10⁶cells/ml). Then, cells were put in 96-well V-bottom plates (100 μl/well)and washed twice in PBS-BSA (PBS supplemented with 0.1% BSA and 0.02%Na-azide). Thereafter, 50 μl antibody solution in PBS-BSA was added tothe cells (4° C., 30 min). After washing three times in PBS-BSA, 50 μl(1:400 dilution) of rabbit anti-human IgG-FITC in PBS-BSA was added (4°C. in the dark, 30 min). Cells were washed three times and specificbinding of CD38-antibodies to CHO-CD38 and Daudi-luc cells was detectedby flow cytometry. HuMab-KLH (a human monoclonal antibody against KLH(keyhole limpet haemocyanin) generated by Genmab B.V., Utrecht, TheNetherlands by use of the immunization protocols described elsewhereherein) was used as a control. FIGS. 1 and 2 show that -003, -005, and-024 bind to CHO-CD38 cells and to Daudi-luc cells, albeit withdifferent EC₅₀ (Table 1). No binding to control CHO cells is observed(data not shown).

Fresh MM tumor cells were obtained from Dr. Lokhorst (University MedicalCenter Utrecht, Utrecht, The Netherlands. Tumor cells were isolated frombone marrow of multiple myeloma patients by Ficoll (Bio Whittaker;lymphocyte separation medium, cat 17-829E) gradient centrifugation.After harvesting and counting, MM cells (100,000 cells/well) wereresuspended with 25 μl FITC-labeled CD38-specific antibodies and 25 μlCD138. After incubation (4° C., 30 min), cells were washed in PBS-BSAand PE-labeled goat-anti-mouse IgG (1:200; Jackson ImmunoResearch EuropeLtd. Soham, UK) was added. After incubation (4° C., 30 min) and washingof the cells in PBS-BSA, fluorescence was measured by flow cytometry.

FIG. 3 shows that -003, -005 and -024 bind to MM cells.

TABLE 1 EC₅₀ values of binding of anti CD38-antibodies on CHO-CD38cells, Daudi-luc cells and fresh MM tumor cells. CD38-specific EC₅₀CHO-CD38 EC₅₀ Daudi-luc EC₅₀ MM cells antibodies (μg/ml) (μg/ml) (μg/ml)−003 0.54 0.26 0.56 −005 0.23 0.09 0.04 −024 0.08 0.05 0.02

Example 5 Antibody-Dependent Cell-Mediated Cytotoxicity

Daudi-luc cells, fresh multiple myeloma tumor cells, fresh Plasma CellLeukemia tumor cells and JK6L and AMO-1 multiple myeloma cells werecollected (5×10⁶ cells) in RPMI++(RPMI 1640 culture medium supplementedwith 10% cosmic calf serum (HyClone, Logan, Utah, USA)), to which 100ηCi ⁵¹Cr (Chromium-51; Amersham Biosciences Europe GmbH, Roosendaal, TheNetherlands) was added, and the mixture was incubated in a 37° C. waterbath for 1 hr. After washing of the cells (twice in PBS, 1500 rpm, 5min), the cells were resuspended in RPMI++ and counted by trypan blueexclusion. Cells were brought at concentration of 1×10⁵ cells/ml.

Preparation of Effector Cells

Fresh peripheral blood mononuclear cells (healthy volunteers, UMCUtrecht, Utrecht, The Netherlands) were isolated from 40 ml of heparinblood by Ficoll (Bio Whittaker; lymphocyte separation medium, cat17-829E) according to the manufacturer's instructions. Afterresuspension of cells in RPMI^(++,) cells were counted by trypan blueexclusion and brought at concentration of 1×10⁷ cells/ml.

ADCC Set Up

50 μl of ⁵¹Cr-labeled targets cells were pipetted into 96-well plates,and 50 μl of antibody was added, diluted in RPMI++(final concentrations10, 1, 0.1, 0.01 μg/ml). Cells were incubated (RT, 15 min), and 50 μleffector cells were added, resulting in an effector to target ratio of100:1 (for determination of maximal lysis, 100 μl 5% Triton-X100 wasadded instead of effector cells; for determination of spontaneous lysis,50 μl target cells and 100 μl RPMI++ were used). Cells were spun down(500 rpm, 5 min), and incubated (37° C., 5% CO₂, 4 hr). After spinningdown cells (1500 rpm, 5 min), 100 μl of supernatant was harvested intomicronic tubes, and counted in gamma counter. The percentage specificlysis was calculated as follows:

(cpm sample−cpm target cells only)/(cpm maximal lysis−cpm target cellsonly) wherein cpm is counts per minute.

In Daudi-luc cells (FIG. 4 and Table 2) -003, -005, and -024 inducelysis by ADCC, and -003, and -005 perform slightly better than rituximab(anti-CD20 mAb). Interestingly, also when fresh multiple myeloma tumorcells (obtained from Dr. H. Lokhorst, UMCU, The Netherlands) are used astarget cells, ADCC is induced by -003, -005 and -024 (FIG. 5A and Table2).

TABLE 2 EC₅₀ values of CD38-specific antibodies obtained in ADCCCD38-specific EC₅₀ Daudi-luc EC₅₀ MM cells antibodies (ng/ml) (ng/ml)−003 9.0 27 −005 4.5 5.7 −024 9.7 56Enrichment of human peripheral blood mononuclear cells Erlangen

Human blood from human volunteers (university Erlangen, Erlangen,Germany) was diluted twice in RPMI 1640 and blood cells were layered onFicoll (Lymphocyte Separation Medium 1077 g/ml, 710 g, RT, 20 min;BioWhittaker, Cambrex Bio Science Verviers, Verviers, Belgium, cat.17-829E, lot no. 0148 32). Peripheral blood mononuclear cells (MNCs)were collected from the interphase, washed and resuspended in RPMI 1640culture medium supplemented with 10% FCS, 2 mM L-glutamine, 5 U/mlpenicillin, 50 μg/ml streptomycin (all derived from BioWhittaker) towhich 25 mM HEPES (BioWhittaker) was added.

ADCC Set Up II

Target B-cells (fresh plasma cell leukemia tumor cells, JK6L and AMO-1B-cell lines, obtained from Dr. T. Valerius, University of Erlangen,Erlangen, Germany) were labeled with 20 μCi ⁵¹Cr (Amersham Biosciences,Uppsala, Sweden) for 2 hours. After extensive washing in RPMI-10, cellswere adjusted to 1×10⁵ cells/ml. MNCs (50 μl), sensitizing antibodies(50 μl), and RPMI-10 (50 μl) were added to round-bottom microtiterplates (Greiner Bio-One GmbH, Frickenhausen, Germany). Assays werestarted by adding fresh plasma cell leukemia tumor cells, JK6L or AMO-1cells (50 μl) giving a final volume of 200 μl. An effector to target(E:T) ratio of 40:1 was used. After incubation (3 hr, 37° C.), assayswere stopped by centrifugation, and ⁵¹Cr release from triplicates wasmeasured in counts per minute (cpm) in a scintillation counter.

Percentage of cellular cytotoxicity was calculated using the followingformula:

% specific lysis=(experimental cpm−basal cpm)/(maximal cpm−basalcpm)×100

with maximal ⁵¹Cr release determined by adding perchloric acid (3% finalconcentration) to target cells, and basal release was measured in theabsence of sensitizing antibodies and effector cells.

In both multiple myeloma cell lines (i.e. JK6L and AMO-1), lysis isinduced with both -003 and -005 (FIGS. 6 and 7), even when CD38expression is low (AMO-1 cell line).

-003, -005 and -024 induce ADCC of plasma cell leukemia primary tumorcells (FIG. 5B).

Example 6 Complement-Dependent Cytotoxicity

After harvesting and counting of Daudi-luc cells, the viability of thecells should be ≥90%. After washing (PBS), cells are resuspended at2.0×10⁶ cells/ml in RPMI-B (RPMI supplemented with 1% BSA). Thereafter,cells are put in 96-well round-bottom plates at 1×10⁵ cells/well (50μl/well). Then, 50 μl antibodies is added to the wells (finalconcentration range between 0-100 μg/ml (three-fold dilutions inRPMI-B)). After incubation (RT, 15 min), 11 μl of pooled human serum(pool of 18 healthy donors) was added to each well (37° C., 45 min).Wells were resuspended once and 120 μl was transferred to FACS tubes(Greiner). Then, 10 μl propidium iodide (PI; Sigma-Aldrich Chemie B.V.)was added (10 μg/ml solution) to this suspension. Lysis was detected byflow cytometry (FACScalibur™, Becton Dickinson, San Diego, Calif., USA)by measurement of the percentage of dead cells (corresponds toPI-positive cells).

FIG. 8 and Table 2 show that lysis of Daudi-luc cells is induced by -005(˜60% maximum lysis) and that lysis by -003 is only seen at very highantibody concentrations. -024 does not induce CDC in Daudi cells (datanot shown). In CHO-CD38 cells, lysis is induced by both -003, -005, and-024 (FIG. 9 and Table 3). Lysis by -003 is induced at higherconcentrations. In tumor cells (all obtained from Dr. Lokhorst and Dr.Bloem, University Medical Center Utrecht, The Netherlands), obtainedfrom different MM patients (A: 3% refractory tumor cells, B: 9%refractory tumor cells, C: 30-40% tumor cells, and D: 70% tumor cells),CDC-mediated lysis is observed in the presence of -005, but not in thepresence of -003 (FIG. 10). -024 also induced lysis of MM tumor cells(FIG. 10E).

TABLE 3 EC₅₀ values of CD38-specific antibodies obtained in CDCCD38-specific EC₅₀ Daudi-luc EC₅₀ CD38-CHO antibodies (μg/ml) (μg/ml)−003 >90 3.14 −005 0.33 0.14 −024 >90 0.24

Example 7

Cross-Block Studies Using FACS CHO-CD38 cells were incubated with anexcess of unlabelled CD38-specific antibody (4° C., 15 min). Then, cellswere incubated with FITC-labeled CD38-specific antibodies (concentrationapproximates EC₉₀, 4° C., 45 min). After twice washing the cells withPBS-BSA, fluorescence was measured by flow cytometry. FIG. 11 shows thatunlabelled -003 blocks binding of FITC-labeled -003, whereas binding ofFITC-labeled -005 is not blocked. Also unlabelled -005 blocks binding ofFITC-labeled -005, whereas binding of FITC-labeled -003 is not blocked.-003 and -005 bind to different epitopes, because they do not competefor binding.

Example 8 Cross-Blocking Studies Using ELISA

Soluble human CD38 is coated on the surface of an ELISA plate. CoatedCD38 is incubated with an excess of unlabelled CD38 specific antibodiesfor about 15 minutes and subsequently biotinylated CD38-specificantibodies are added (concentration approximates EC₉₀, RT, 1 hour).After washing three times with PBS/Tween, horseradish peroxidase(HRP)-conjugated streptavidine is added and the mixture is incubated for1 hour at RT. The complex can be detected by addition of anABTS-solution and the HRP mediated substrate conversion is measuredusing an ELISA reader at OD 405 nm.

Example 9 Cross-Blocking Studies Using Sandwich-ELISA

CD38 specific antibodies are coated on the surface of an ELISA plate.Plate-bound antibodies are incubated with biotinylated soluble CD38 inthe presence of an excess of CD38 specific antibodies in fluid phase.After washing with PBS/Tween, bound biotinylated CD38 is detected withHRP-conjugated streptavidine for 1 hr at RT. This complex can bedetected by addition of an ABTS-solution (after washing with PBS/Tween)and the HRP mediated substrate conversion is measured using an ELISAreader at OD 405 nm.

Example 10

Reactivity with a Panel of Human Tissues and Cross-Reactivity withCynomolgus Tissue by Immunohistochemistry

Sections from frozen human tissue (obtained from Dr. H. Niessen, FreeUniversity Medical Center, Amsterdam, The Netherlands) or monkey tissue(Inveresk Research, Glasgow, Scotland) were cut at 6 μm and air-driedovernight. These cryostat sections were fixated in acetone (RT, 10 min)and air-dried (approx. 5 min). Thereafter, sections were incubated with1× citric acid/phosphate buffer containing 0.1% H₂O₂(pH 5.8; Sigma), toblock endogenous peroxidase. After 20 min at RT, sections were washedtwice with PBS and 0.05% Tween-20 (PBST, RT, 5 min; Riedel de-Haen,Germany). Then, sections were incubated with avidin (RT, 15 min; DAKO,Glostrup, Denmark), washed twice with PBST, and incubated with biotin(RT, 15 min; DAKO) to block endogenous biotin. After washing thesections twice with PBST, sections were pre-incubated with PBST⁺⁺ (PBSTsupplemented with 10% normal human serum (NHS, CLB, Amsterdam,Netherlands) and 10% normal goat serum (NGS; DAKO) (RT, 20 min). Afterblotting-off of the pre-incubation serum, sections were incubated withFITC-labeled primary antibody diluted in 2% PBST⁺⁺ at the indicatedconcentrations (RT, 60 min). Thereafter, sections were incubated withrabbit-anti-FITC (1:1000; DAKO) in 2% PBST⁺⁺ (RT, 30 min). After washingthe sections with PBST, sections were incubated withgoat-anti-rabbit-biotin (1:400; DAKO) in 2% PBST⁺⁺ (RT, 30 min). Then,sections were washed and incubated with SABC-HRP (1:100; DAKO) in 2%PBST⁺⁺ (RT, 30 min). After washing the sections twice in PBST, they wereincubated (RT, 10 min) with amino-ethyl-carbazole (AEC)-developmentsolution (50 mM acetate buffer, pH4.9, 0.01% H₂O₂; Riedel-de-Haen).Finally, sections were washed in millipore H₂O (5 min) andcounterstained with hematoxylin (DAKO). By use of glycergel (37° C.),sections were fixed with cover slips, and studied by light microscopy(Axiovision-2; Zeiss, Thornwood, N.Y., USA).

Bronchial epithelium is stained with -003 and -005 (FIGS. 12B and 13B)as well as striated muscle (myocytes, FIGS. 12C and 13C), macrophages,lymphocytes and plasma B cells (FIGS. 12A and 13A). -024 has a similarstaining of striated muscle and bronchial epithelium, but staining wasless intense. No staining of endothelial cells is observed, neither with-003 (FIG. 14D), -005 (14E) nor -024 (data not shown), whereas clearstaining was observed with the positive control antibodies againstendothelial cell markers CD31 (FIG. 14A) and vWF (14B). Anti-KLH wasused as negative control antibody (FIG. 14C). -003 (FIG. 12D) and -024(data not shown) but not -005 (FIG. 13D) cross-react with cynomolgusmonkey lymphoid tissue.

Example 11

Cross-Reactivity with Cynomolgus or Rhesus Monkey Peripheral BloodMononuclear Cells (PBMCs) by Flow Cytometry

5 ml of cynomolgus monkey peripheral blood (Inveresk Research) werelysed by adding 4.5 ml shock buffer (1.7 mM NH4CL, 1 mM EDTA), 40 ml H₂Oand 450 μl 10% KHCO₃. After hemolysis cells were centrifuged (1200 rpm,10 min) and washed thrice in PBS. After counting cells with trypan blue,cells were resuspended in PBS-BSA (1×10⁶ cell/ml).

17.5 ml of rhesus monkey peripheral blood (BPRC, Rijswijk, TheNetherlands) was diluted 1:1 with RPMI 1640 and layered on Ficoll (1.077g/ml; BioWhittaker, cat. 17-829E, lot no. 0148 32). After centrifugation(710 g, RT, 20 min), the interphase was collected and washed twice inRPMI. After the last wash cells were resuspended in RPMI 1640 at aconcentration of 1×10⁵ cells/50 μl.

Cells were transferred to 96-well plate (100,000 PBMCs/well), washed inFACS buffer (PBS, 0.05% BSA, 0.02% NaN₃) and incubated with the primaryantibodies (4° C., 30 min). After washing in PBS-BSA, 50 μl FITC-labeledrb-anti-hIgG (DAKO, Glostrup, Denmark) was added (4° C., 30 min).Finally, cells were collected in FACS tubes in a total volume of 150 μl.Samples were measured and analyzed by use of FACScalibur™ (BectonDickinson, San Diego, Calif., USA).

With flow cytometry cross-reactivity of -003 on cynomolgus lymphocytes(FIG. 15A) and monocytes (FIG. 15B) was shown, but not of -005. Also inrhesus monkeys, cross-reactivity of -003 was observed on mononuclearcells, but not of -005 (FIG. 15C).

Example 12 Internalization Experiments

CHO-CD38 cells were stained with a saturating concentration ofFITC-labeled CD38-specific antibodies (on ice, 30 min). After washing ofcells (in RPMI1640 supplemented with 10% FCS), one cell pool was warmedup to 37° C. to allow internalization, and the other pool was left onice. At several time intervals (0-120 min) cell aliquots were taken andtransferred to ice-cold PBS-BSA to stop internalization. After washingsamples twice with PBS-BSA, EtBr (diluted in PBS-BSA, finalconcentration 2 mg/ml) was added to the samples to quench membrane-boundFITC. Fluorescence was measured by flow cytometry.

FIGS. 16A and 16B show that -003 and -005 are internalized by CHO-CD38cells within 5 minutes at 37° C.

Example 13 In Vivo SCID-Luciferase Experiments

In this model tumor cells are transfected with firefly luciferase. Uponadministration of luciferin (Molecular Probes, Leiden, The Netherlands)to the mice the labeled cells can be detected in vivo by bioluminescentimaging using a highly sensitive CCD camera, cf. Wetterwald et al.,American Journal of Pathology 160(3), 1143-1153 (2002).

Daudi cells were transfected with gWIZ luciferase from Gene TherapySystems (San Diego, Calif.) and cultured in RPMI with 10% FCS,Pen/Strep, Sodium Pyruvate and 1 μg/ml puromycin (Sigma). Cells wereanalysed for luciferase expression (expressed in RLU/1×105 cells) in aluminometer and for CD38 expression by FACS. 2.5×10⁶luciferase-transfected Daudi cells/mouse were injected i.v. into SCIDmice. Mice were treated with -003, -005, isotype control antibody(HuMab-KLH) or rituximab (anti-CD20 antibody). Antibodies were injectedintraperitoneally. Four treatment settings were used (see Table 4). Inthe preventive setting, antibody (100 μg/mouse) and cells wereadministered simultaneously. In therapeutic setting I, antibody (300μg/mouse) was administered 7 days after administration of cells. Intherapeutic setting II, antibody (10 μg/mouse) was administered 14 daysafter administration of cells. In therapeutic setting III, antibody (100μg/mouse) was administered 7 days after administration of cells. Forimaging, mice were anesthetized by i.p. injection of a mixture ofketamine/xylazine/atropine. Synthetic D-Luciferin (sodium salt,Molecular Probes) was given i.p. at a dose of 25 mg/ml. Mice were thenplaced in a light tight box and after 3 min, imaging was started using aVersArray 1300B liquid nitrogen cooled CCD detector (Roper Scientific).Photons emitted from the luciferase were counted over an exposure periodof 5 min. Under illumination black and white images were made forreference. MetaVue software (Universal Imaging Corp) was used for datacollection and image analysis. Statistical significance of differencesbetween groups was established using one-way analysis of variance with aNewman-Keuls post test using GraphPad PRISM version 3.02 (GraphpadSoftware Inc).

TABLE 4 Treatment settings for in vivo luciferase experiments Antibodytreatment Antibody dose Experimental setting (days after cellinoculation) (μg/mouse) Preventive setting 0 100 Therapeutic setting 7300 Therapeutic setting II 14 10 Therapeutic setting III 7 100

FIGS. 17A and 17B show that -003 and -005 inhibit growth of tumor cellsin the preventive setting and in therapeutic setting I, similar to theinhibition observed for the anti-CD20 antibody. Both antibodies performsignificantly better than the isotype control antibody. Also intherapeutic setting II CD38-antibodies slow down the growth of Daudi-luctumor cells (FIG. 17C). In therapeutic setting III, -003 and -024 show aclear inhibition of Daudi-luc tumor cell growth (FIG. 17D).

Example 14 Apoptosis

Apoptosis assay was carried out according to the manufacturer'sinstructions (Annexin-V Apoptosis kit, BD Biosciences, Alphen a.d. Rijn,Netherlands). In short, CD38 mAbs were added to 2.5×10⁵ cells(luciferase-transfected Daudi cells, in 0.5 ml RPMI⁺⁺ in a 24-wellsplate), in a concentration of 5 μg/ml -003 or -005 or an anti-CD20antibodies alone or in the presence of cross-blocking rb-anti-hlgG (50μg/ml).

After incubation (37° C., 5% CO₂, 20 hr), cells were harvestedcarefully, and washed with Binding Buffer (1200 rpm, 4° C., 5 min, BDBiosciences). Pellet was resuspended in 100 μl Binding Buffer. Then, 5μl Annexin-V-FITC (BD Biosciences) and 10 μl PI (BD Biosciences) wasadded to the suspension and incubated for 15 minutes at RT. 400 μlBinding Buffer was added and the samples were measured (PI readout inFL2). For analysis of apoptotic cells, all Annexin-V-positive cells werecounted by flow cytometry using a FACScalibur flow cytometer withCellQuest pro software (BD Biosciences). At least 10,000 events werecollected for analysis. This population includes both PI-positive aswell as PI-negative cells.

FIG. 18 shows that -003 and -005 do not induce apoptosis. However, aftercross-linking, apoptosis of target cells is observed. -003 inducedapoptosis after cross-linking that was similar to apoptosis induced byan anti-CD20 antibody (rituximab). -005 was less able to induceapoptosis after cross-linking. Similar results were obtained with RAMOScells as target cells (data not shown).

Example 15 Effect of -005 on Tissue Graft B Cells in RA-SCID Mouse Model

Implantation of Synovial Tissue

SCID-mice, strain C.B.-17/lcrCrl-SCID-bg, male/female, 4-12 weeks,purchased from Charles River Laboratories Nederland (Maastricht, theNetherlands) were kept in IVC cages under standard conditions oftemperature and light, and were fed laboratory chow and water adlibitum. Prior to implantation, mice (three mice in each experimentalgroup, day 0) were anesthetized by intraperitoneal injection of ketamine(NIMATEK, EuroVet) and xylazine (Rompun, Bayer) at ratio 1:1. A smallincision of the skin was made using surgical scissors. Inflamed synovialtissue from a patient with rheumatoid arthritis undergoing jointreplacement surgery was implanted subcutaneously as a cluster of sixsmall fragments (total 2-3 mm³) on each flank of the mouse. The woundwas closed using Permacol cyanoacrylate glue. On day 1 of theexperiment, remaining synovial tissue was analyzed in order to check forB cells in the inflamed synovial transplants. -005 (12 mg/kg) or controlantibody (anti-KLH, 30 mg/kg) was injected (i.v.), in a volume of 200 μlon day 8 of the experiment. At the end of the experiment (day 14) micewere sacrificed by CO₂ inhalation and the synovial grafts wereexplanted. One of the grafts was snap-frozen in OCT compound (TissueTek,Sacura Finetek Europe) for further immunhistochemical analysis, andanother one was frozen by immersion in liquid nitrogen for further RNAanalysis.

Immunohistochemistry

5 μM cryosections on SuperFrost (Menzel GmbH, Braunschweig) slides wereprepared using LEICA CM1900 cryostate and stored at −80° C. Thawedsections were fixed in acetone for 10 min, dried at room temperature andwashed 3×5 min in PBS. All steps were performed at room temperature.Endogenous peroxidase activity was blocked by incubation with PBSsupplemented with 0.3% hydrogen peroxide and 0.1% sodium azide for 20min. Slides were washed 3×5 min in PBS and incubated with 10% normalhuman serum (NHS)/10% normal rabbit serum (NRbS) in PBS/1% BSA for 30min. Next, primary antibody (mouse mAb) diluted in PBS supplemented with1% BSA/10% NHS/10% NRbS was incubated for 60 min. After 3×2 min washesin PBS, HRP-conjugate (goat anti-mouse Ig-HRP; DAKO P0447) diluted 1:50in PBS (supplemented with 1% BSA/10% NHS/10% NRbS) was added for 30 min.Peroxidase signal was enhanced using TSA™ Biotin system (Perkin ElmerLife Sciences, NEL700). Slides were washed 3×2 min in PBS and incubatedwith biotinyl tyramide diluted 1:1600 in amplification buffer for 30min. After 3×2 min washes in PBS, streptavidin-HRP diluted 1:400 in PBS(supplemented with 1% BSA) was added for 30 min. Slides were washed 3×2min in PBS and incubated with DAB solution (DAKO Cytomation K3465) for 5min. Color reaction was stopped with distilled water. Finally, slideswere counterstained with hematoxyline (MERCK), washed with running waterand covered with Kaiser's glycerin and cover slips.

Scoring of Staining Intensity

Scoring of stained synovial tissue xenografts was performed in a blindedfashion by two trained persons. First the strongest section was selectedfrom a series of sections and this reference section was awarded themaximum score 8. The staining intensity in the other sections was thenscored on a scale of 0 to 8, relative to the reference section.

Statistical Analysis

Scoring of staining intensity was analyzed by Kruskal-Wallis one-wayANOVA followed by Dunn's multiple comparison test using Graph Pad Prismversion 4.01 (Graph Pad software, Inc., San Diego, Calif., USA).

FIG. 19 and FIG. 21 show that the numbers of anti-CD38-positive plasmacells are reduced after treatment with -005. Staining of plasma cellswith anti-CD138 confirms that -005 results in reduced numbers of plasmacells (FIGS. 20 and 22).

Example 16 Sequencing of the Coding Sequence of Human Antibodies AgainstCD38

RNA Preparation

Total RNA was prepared from 5×10⁶ cells of the hybridoma cell linesexpressing the monoclonal antibody -003, -005 and -024, respectively,with the RNeasy kit (Qiagen, Westburg, Leusden, Netherlands) accordingto the manufacturer's protocol.

cDNA Preparation of -003, -005 and -024

5′-RACE-Complementary DNA (cDNA) of RNA was prepared from 100 ng totalRNA, using the SMART RACE cDNA Amplification kit (Clontech), followingthe manufacturer's protocol.

Oligonucleotide primers were synthesized and quantified by IsogenBioscience (Maarssen, The Netherlands). Primers were dissolved in H₂O to100 pmol/μl and stored at −20° C. A summary of all PCR and sequencingprimers is tabulated (Table 5). For PCR, PfuTurbo® Hotstart DNApolymerase (Stratagene, Amsterdam, The Netherlands; product #600322) wasused according to the manufacturer's instructions. Each reaction mixcontained 200 μM mixed dNTPs (Roche Diagnostics, Almere, TheNetherlands; product #1814362), 12 μmol of the reverse primer (RACEG1A1for V_(H)3003-005, RACEV_(H)ApaI for V_(H)3003-003 and RACEV_(L)BsiWIfor V_(L)3003-003 and 005), 7.2 pmol UPM-Mix (UPM-Mix: 2 μM ShortUPMH3and 0.4 μM LongUPMH3), 0.6 μl of the 5′RACE cDNA template, and 1.5 unitof PfuTurbo® Hotstart DNA polymerase in PCR reaction buffer (suppliedwith polymerase) in a total volume of 30 μl. PCR reactions were carriedout with a TGradient Thermocycler 96 (Whatman Biometra, Goettingen,Germany; product #050-801) using a 35-cycle program: denaturing at 95°C. for 2 min; 35 cycles of 95° C. for 30 sec, a 55° C. for 30 sec, and72° C. for 1.5 min; final extension at 72° C. for 10 min. Ifappropriate, the PCR mixes were stored at 4° C. until further analysisor processing.

TABLE 5 Primers Name Sequence ShortUPMH3 TGAAAGCTTCTAATACGACTCACTATAGGGCRACEV_(L)BsiWi GAAGATGAAGACAGATGGTGCAGCCACCGTACG RACEV_(H)ApaIGGAGGGTGCCAGGGGGAAGACCGATGGGCCCTT RACEG1A1 GGGAGTAGAGTCCTGAGGACTGM13reverse GGATAACAATTTCACACAGG Long UPMH3TGAAAGCTTCTAATACGACTCACTATAGGGCAAGC AGTGGTATCAACGCAGAGT HCseq5GGTCAGGGCGCCTGAGTTCCACG VH3003-003forGATAAGCTTGCCGCCACCATGGACTGGACCTGGAG GTTCCTC VH3003-5forGATAAGCTTGCCGCCACCATGGAGTTTGGGCTGAG CTGGCTT VL3003-5exforGATAAGCTTGCCGCCACCATGGAAGCCCCAGCTCA GCTTCTC VL3003-003forGATAAGCTTGCCGCCACCATGAGGGTCCTCGCTCA GCTCCTG VH300324exforGATAAGCTTGCCGCCACCATGGGGTCAACCGCCAT CCTCGCC VL3003-24-GATAAGCTTGCCGCCACCATGGAAGCCCCAGCTCA 5exfor GCTTCTC

Cloning of -003-2F5 V_(H) and V_(L) and -005 V_(L) and -024 V_(H) andV_(L) in pGEMT-Vector System II

The reaction products were separated by electrophoresis on a 1% TAEagarose gel and stained with ethidium bromide. Bands of the correct sizewere cut from the gels and the DNA was isolated from the agarose usingthe Qiaexll gel extraction kit (Qiagen, cat no 20021).

Gel isolated PCR fragments were A tailed by a 10 min 72° C. incubationwith 200 μM dATP and 2.5 units Amplitaq (Perkin Elmer) and purifiedusing minielute columns (Qiagen). A-tailed PCR fragments were clonedinto the pGEMTeasy vector (Promega) using the pGEMT easy vector systemII kit and protocol (LJ270, page 3/4). 2 μl of the ligation mixture wastransformed into OneShot DH5αT1R competent E. Coli (Invitrogen) andplated on LB/Amp/IPTG/Xgal plates.

Sequencing

The V-regions -003 and -024 and the -005 V_(L) region were sequenced byAGOWA (Berlin, Germany) after picking respectively 20 (V_(H)-003), 16(V_(L)-003), 15 (V_(L)-005) and 6 (VH and VL -024) white colonies,isolating plasmid and sequencing with the M13 reverse primer. The -005V_(H) region was sequenced directly on the PCR product by using primerHCseq5. Sequences were analyzed using the Vector NTI advanced suite(Invitrogen).

Generation of Expression Vectors for Antibody -003, -005, -024 andMorphosys Antibody 3079

The V_(H) coding region of -003 was amplified by PCR from a pGemTplasmid clone containing the V_(H) region of -003, using the primersVH3003-003 for and RACEVHApaI, introducing suitable restriction sites(HindIII and ApaI) for cloning into pConGlf0.4 (Lonza Biologics, Slough,UK) and an ideal Kozak sequence (GCCGCCACC). The pConGlf0.4 vectorcontains the heavy chain constant region of human IgG1. The V_(H) PCRfragment was inserted, in frame, into the pConGlf0.4 vector usingHindIII and ApaI. The construct was checked by sequence analysis.

The V_(H) coding region of -005 was amplified by PCR from a pGemTplasmid clone containing the V_(H) region of -005, using the primersVH3003-5 for and RACEVHApaI, introducing suitable restriction sites(HindIII and ApaI) for cloning into pConGlf0.4 and an ideal Kozaksequence. The V_(H) PCR fragment was inserted, in frame, into thepConGlf0.4 vector using HindIII and ApaI. The construct was checked bysequence analysis.

The V_(H) coding region of -024 was amplified by PCR from a pGemTplasmid clone containing the V_(H) region of -024, using the primersVH300324exfor and RACEVHApaI, introducing suitable restriction sites(HindIII and ApaI) for cloning into pConGlf0.4 and an ideal Kozaksequence. The V_(H) PCR fragment was inserted, in frame, into thepConGlf0.4 vector using HindIII and ApaI. The construct was checked bysequence analysis.

The V_(H) coding region of Morphosys antibody 3079 was synthesized byGeneArt (Regensburg, Germany), based on the data published in patent WO2005/103083 A2.

The coding region was codon optimized for expression in HEK cells toenhance expression levels and suitable restriction sites (HindIII andApaI) for cloning into pConGlf0.4 and an ideal Kozak sequence wereintroduced. The plasmid containing the synthetic VH region was digestedwith ApaI and HindIII and the VH fragment was inserted, in frame, intothe pConGlf0.4 vector.

The V_(L) coding region of -005 was amplified by PCR from a pGemTplasmid clone containing the V_(L) region of -005, using the primersVL3003-5exfor and RACEVLBsiWI, introducing suitable restriction sites(HindIII and Pf123II) for cloning into pConKappa0.4 (Lonza Biologics)and an ideal Kozak sequence. The pConKappa0.4 vector contains the kappalight chain constant region. The V_(L) PCR fragment was inserted, inframe, into the pConKappa0.4 vector using HindIII and Pf123II. Theconstruct was checked by sequence analysis.

The V_(L) coding region of -003 was amplified by PCR from a pGemTplasmid clone containing the V_(L) region of -003, using the primersVL3003-003 for and RACEVLBsiWI, introducing suitable restriction sites(HindIII and Pf123II) for cloning into pConKappa0.4 and an ideal Kozaksequence. The V_(L) PCR fragment was inserted, in frame, into thepConKappa0.4 vector using HindIII and Pf123II. The construct was checkedby sequence analysis.

The V_(L) coding region of -024 was amplified by PCR from a pGemTplasmid clone containing the V_(L) region of -024, using the primersVL3003-24-5exfor and RACEVLBsiWI, introducing suitable restriction sites(HindIII and Pf123II) for cloning into pConKappa0.4 and an ideal Kozaksequence. The V_(L) PCR fragment was inserted, in frame, into thepConKappa0.4 vector using HindIII and Pf123II. The construct was checkedby sequence analysis.

The V_(L) coding region of Morphosys antibody 3079 was synthesized byGeneArt, based on the data published in WO 2005/103083. The codingregion was codon optimized for expression in HEK cells; to enhanceexpression levels and suitable restriction sites (HindIII and Pf123II)for cloning into pConKappa0.4 and an ideal Kozak sequence wereintroduced. The plasmid, containing the synthetic V_(L) region, wasdigested with Pf123II and HindIII and the VH fragment was inserted, inframe, into the pConKappa0.4 vector.

Antibodies were transiently expressed in HEK-293F cells, as described inExample 17, by cotransfecting their heavy chain and light chain vectors.

Generation of Stable Cell Lines in CHO-K1SV Cells

For generation of stable cell lines, the heavy and light chain vectorsof -003 or -005 were combined in a single double gene vector by standardcloning techniques.

The double gene vectors of -003 or -005 were linearized and transfectedinto CHO-K1SV (Lonza Biologics) cells, essentially as described by themanufacturer. Stable cell lines were selected by selection with 25 μML-Methionine sulphoximine (MSX) as described by Lonza Biologics. Topproducing clones were selected and propagated in CD-CHO (Invitrogen)medium and antibodies were purified from cell culture supernatant asdescribed in Example 3.

Example 17 Epitope Mapping Using Site Directed Mutagenesis

Oligonucleotide primers were synthesized and quantified by IsogenBioscience (Maarssen, The Netherlands). Primers were dissolved in H₂O to100 pmol/μl and stored at −20° C. A summary of all PCR and sequencingprimers is shown in Table 6. For PCR, PfuTurbo® Hotstart DNA polymerase(Stratagene, Amsterdam, The Netherlands) was used according to themanufacturer's instructions. Each reaction mix contained 200 μM mixeddNTPs (Roche Diagnostics, Almere, The Netherlands), 10 pmol of both theforward and reverse primer, 100 ng of genomic DNA or 1 ng of plasmid DNAand 1 unit of PfuTurbo® Hotstart DNA polymerase in PCR reaction buffer(supplied with polymerase) in a total volume of 20 μl. PCR reactionswere carried out with a TGradient Thermocycler 96 (Whatman Biometra,Goettingen, Germany) using a 32-cycle program: denaturing at 95° C. for2 min; 30 cycles of 95° C. for 30 sec, a 60-70° C. gradient (or anotherspecific annealing temperature) for 30 sec, and 72° C. for 3 min; finalextension at 72° C. for 10 min. If appropriate, the PCR mixtures werestored at 4° C. until further analysis or processing.

Agarose gel electrophoresis was performed according to Sambrook(Sambrook, Russell et al. 2000) using gels of 50 ml, in 1× Tris AcetateEDTA buffer. DNA was visualized by the inclusion of ethidium bromide inthe gel and observation under UV light. Gel images were recorded by aCCD camera and an image analysis system (GeneGnome; Syngene, viaWestburg B.V., Leusden, The Netherlands).

Purification of desired PCR fragments was carried out using a MinElutePCR Purification Kit (Qiagen, via Westburg, Leusden, The Netherlands;product #28006), according to the manufacturer's instructions. IsolatedDNA was quantified by UV spectroscopy (see below) and the quality wasassessed by agarose gel electrophoresis.

Alternatively, PCR or digestion products were separated by agarose gelelectrophoresis (for instance when multiple fragments were present)using a 1% Tris Acetate EDTA agarose gel. The desired fragment wasexcised from the gel and recovered using the QIAEX II Gel Extraction Kit(Qiagen; product #20051), according to the manufacturer's instructions.

Optical density of nucleic acids was determined using a NanoDrop ND-1000Spectrophotometer (Isogen Life Science, Maarssen, The Netherlands)according to the manufacturer's instructions. The DNA concentration wasmeasured by analysis of the optical density (OD) at 260 nm (oneOD_(260 nm) unit=50 μg/ml). For all samples, the buffer in which thenucleic acids were dissolved was used as a reference.

Restriction enzymes and supplements were obtained from New EnglandBiolabs (Beverly, Mass., USA) or Fermetas (Vilnius, Lithuania) and usedaccording to the manufacturer's instructions. DNA (100 ng) was digestedwith 5 units of enzyme(s) in the appropriate buffer in a final volume of10 μl (reaction volumes were scaled up as appropriate). Digestions wereincubated at the recommended temperature for a minimum of 60 min. Forfragments requiring double digestions with restriction enzymes whichinvolve incompatible buffers or temperature requirements, digestionswere performed sequentially. If necessary digestion products werepurified by agarose gel electrophoresis and gel extraction.

Ligations of DNA fragments were performed with the Quick Ligation Kit(New England Biolabs) according to the manufacturer's instructions. Foreach ligation, vector DNA was mixed with approximately three-fold molarexcess of insert DNA.

Plasmid DNA (1-5 μl of DNA solution, typically 2 μl of DNA ligation mix)was transformed into One Shot DH5α-T1^(R) E. coli cells (Invitrogen,Breda, The Netherlands; product #12297-016) using the heat-shock method,according to the manufacturer's instructions. Next, cells were plated onLuria-Bertani (LB) agar plates containing 50 μg/ml ampicillin. Plateswere incubated for 16-18 h at 37° C. until bacterial colonies becameevident.

Bacterial colonies were screened for the presence of vectors containingthe desired sequences via colony PCR using the ThermoStart PCR MasterMix (Abgene, via Wetsburg, Leusden, The Netherlands; product #AB-938-DC15/b) and primers pConG1seq1 and pEE13.4seqrev2 (Table 6).Selected colonies were lightly touched with a 20 μl pipette tip andtouched briefly in 2 ml LB for small scale culture, and then resuspendedin the PCR mix. PCR was performed with a TGradient Thermocycler 96 usinga 35-cycle program: denaturation at 95° C. for 15 min; 35 cycles of 94°C. for 30 sec, 55° C. for 30 sec and 72° C. for 2 min; followed by afinal extension step of 10 min at 72° C. If appropriate, the PCRmixtures were stored at 4° C. until analysis by agarose gelelectrophoresis.

Plasmid DNA was isolated from E. coli cultures using the following kitsfrom Qiagen (via Westburg, Leusden, The Netherlands), according to themanufacturer's instructions. For bulk plasmid preparation (50-150 mlculture), either a HiSpeed Plasmid Maxi Kit (product #12663) or aHiSpeed Plasmid Midi Kit (product #12643) was used. For small scaleplasmid preparation (±2 ml culture) a Qiaprep Spin Miniprep Kit (product#27106) was used and DNA was eluted in 50 μl elution buffer (suppliedwith kit).

Construction of HA-CD38 Expression Vector pEE13.4HACD38

The extracellular domain of human CD38 was amplified from plasmidpClpuroCD38 (obtained from Prof. M. Glennie, Tenovus ResearchLaboratory, Southampton General Hospital, Southampton, UK) using primerscd38forha and cd38exrev. By this PCR reaction an HA-tag was introduced.This PCR product was used as template for a second PCR reaction withprimers SPHMM38ex and cd38exrev. By this PCR reaction, signal peptideSPHMM, restriction sites and an ideal Kozak sequence (GCCGCCACC) foroptimal expression were introduced. After purification, this PCRfragment was cloned into expression vector pEE13.4 (Lonza Biologics) andthe complete coding sequence was confirmed by sequencing with primerspConKseql, pEE13.4seqrev, cd38seqlfor and cd38seq2rev (Table 6). Thisconstruct was named pEE13.4HACD38

Site-Directed Mutaqenesis

Three single mutant proteins of huCD38 was constructed, in which T wasmutated to A at position 237 (T237A, SEQ ID No:32), Q was mutated to Rat position 272 (Q272R, SEQ ID No:33), or S was mutated to F at position274 (S274F, SEQ ID No:34). Site-directed mutagenesis was performed usingthe QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene,Amsterdam, The Netherlands) according to the manufacturer'sinstructions. This method included the introduction of a silent extrarestriction site or loss of a restriction site to screen for successfulmutagenesis (extra Xba1 site for T237A mutant, extra Bcg1 site for Q272Rmutant and loss of Ssp1 site for S274F mutant). Briefly, 5 μl 10×reaction buffer, 1 μl oligonucleotide HACD38T237Afor2, HACD38Q272Rfor orHACD38S274Ffor (100 pmol/μl), 1 μl oligonucleotide HACD38T237Arev2,HACD38Q272Rrev or HACD38S274Frev (100 pmol/μl), 1 μl dNTP mix, 3 μlQuicksolution, 1 μl plasmid pEE13.4HACD38 (50 ng/μl) and 1 μl PfuUltraHF DNA polymerase were mixed in a total volume of 50 μl and amplifiedwith a TGradient Thermocycler 96 (Whatman Biometra, Goettingen, Germany;product #050-801) using an 18-cycle program: denaturing at 95° C. for 1min; 18 cycles of 95° C. for 50 sec, 60° C. for 50 sec, and 68° C. for10 min. PCR mixtures were stored at 4° C. until further processing.Next, PCR mixtures were incubated with 1 μl DpnI for 60 min at 37° C. todigest the pEE13.4HACD38 WT vector and stored at 4° C. until furtherprocessing. The reaction mixture was precipitated with 5 μl 3 M NaAc and125 μl ethanol, incubated for 20 minutes at −20° C. and spun down for 20minutes at 4° C. at 14000×g. The DNA pellet was washed with 70% ethanol,dried and dissolved in 4 μl water. The total 4 μl reaction volume wastransformed in One Shot Top 10DH5α T1^(R) competent E. coli cells(Invitrogen, Breda, The Netherlands) according to the manufacturer'sinstructions (Invitrogen). Next, cells were plated on Luria-Bertani (LB)agar plates containing 50 μg/ml ampicillin. Plates were incubated for16-18 h at 37° C. until bacterial colonies became evident. Colonies werescreened by colony PCR using primers pConG1 seq1 and pEE13.4seqrev2(Table 5) and digested with the relevant restriction enzymes to screenfor incorporation of the mutagenic oligonucleotide. 2 positive clonesfor each mutant were grown and plasmid DNA was isolated. The completeHACD38 coding sequence was determined using primers cd38seq1for,pConG1seq1 and pEE13.4seqrev2 to confirm the presence of the mutationsand the absence of additional undesirable mutations.

DNA Sequencing

Plasmid DNA samples were sent to AGOWA (Berlin, Germany) for sequenceanalysis. Sequences were analyzed using Vector NTI advanced software(Informax, Oxford, UK).

Transient Expression in HEK-293F Cells

Freestyle™ 293-F (a HEK-293 subclone adapted to suspension growth andchemically defined Freestyle medium, (HEK-293F)) cells were obtainedfrom Invitrogen and transfected with pEE13.4HACD38 and with the threeconstructs carrying the mutations T237A, Q272R and S274F, according tothe manufacturer's protocol using 293fectin (Invitrogen). Culturesupernatants of transfected cells were used in ELISA for anti-CD38binding studies.

Anti-CD38 Antibody Binding

ELISA plates (Greiner, #655092) were coated O/N at 4° C. with 1 μganti-HA antibody (Sigma, # H-9658) and subsequently blocked with 2%chicken serum. Culture supernatants of transfected HEK293F cells werediluted, applied to the ELISA plates and incubated for 1 hr at RT. Afterwashing, serial dilutions of HuMabs -003 and -005 were added andincubated for 1 hr at RT. Bound antibodies were detected withHRP-conjugated goat-anti-human IgG antibodies. The assay was developedwith ABTS (Roche, #1112597) and the absorbance was measured at 405 nmusing a spectrophotometer.

As can been seen from FIGS. 23A-23C, both -003 and -005 bind to wt humanCD38. The binding of -003 was not affected by the introduction ofmutations T237A (FIG. 23A), Q272R (FIG. 23B) or S274F (FIG. 23C). -005was able to bind CD38 harboring mutation T237A (FIG. 23A). Binding of-005 to CD38 with mutation Q272R was severely affected (FIG. 23B), bothwith respect to EC₅₀ and maximum binding capacity. -005 was not able tobind to mutant CD38 wherein serine at position 274 was replaced byphenylalanine (FIG. 23C).

These data shows that -003 and -005 bind to different epitopes.Furthermore these studies revealed that binding of -005 to CD38 issensitive to mutations at positions 272 and 274. Particularly S274 isessential for -005 binding to CD38.

TABLE 6 Primers Name Sequence cd38forhaCTGCTGTGGCCCATGGTGTGGGCCTACCCTTAC GACGTGCCTGACTACGCCAGGTGGCGCCAGACGTGGAGC cd38exrev AGGTCAGGTACCTCAGATCTCAGATGTGCAAG SPHMM38exTATAGCCCGGGGCCGCCACCATGTGGTGGCGCC TGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCC pConG1seq1 GAAGACTTAAGGCAGCGGCAGAA pConKseq1GTAGTCTGAGCAGTACTCGTTGC pEE13.4seqrev TGCATTCATTTTATGTTTCAGGTpEE13.4seqrev2 TCGGACATCTCATGACTTTCTTT cd38seq1forAGGACACGCTGCTAGGCTACCTT cd38seq2rev GTCCTTTCTCCAGTCTGGGCAAGHACD38T237Arev2 TCCACCATGTATCACCCAGGCCTCTAGAGCCTG AACCTTCTCTGGTTGHACD38T237Afor2 CAACCAGAGAAGGTTCAGGCTCTAGAGGCCTGG GTGATACATGGTGGAHACD38Q272Rrev GATATTCTTGCAGGAAAATCGAATATTCCTTTT GCTTAT HACD38Q272RforATAAGCAAAAGGAATATTCGATTTTCCTGCAAG AATATC HACD38S274FrevTCTGTAGATATTCTTGCAGAAAAATTGAATGTT CCTTTTGCTTATA HACD38S274FforTATAAGCAAAAGGAACATTCAATTTTTCTGCAA GAATATCTACAGA

Example 18 Induction of Proliferation of PBMC

-003, -005 and -024 were tested in an assay essentially as described inAusiello et al., Tissue antigens 56, 538-547 (2000). Briefly, PBMCs fromhealthy donors were cultured at 1×10⁵ cells/well in flat bottom 96-wellplates in the presence of antibodies (final concentration: 1.1-3.3-10-30μg/ml) in 200 μl RPMI++. Stimulation of cells with IL-15 (at 333 ng/ml;Amgen Inc., Thousand Oaks, Calif., USA) was used as positive control.After a 4 day incubation at 37° C., 30 μl ³H-thymidine (16.7 μCi/ml) wasadded, and culture was continued O/N. ³H-thymidine incorporation wasassessed using a Packard Cobra gamma counter (Packard Instruments,Meriden, DT, USA), according to the manufacturer's instructions. Dataare shown as the mean cpm (±SEM) of PBMCs obtained from 10 donors. Theresults show that -003 and -005 do not induce significant proliferationof PBMCs (FIG. 24A). Also -024 did not induce significant proliferationof PBMCs (data not shown).

Example 19

Induction of IL-6

-003, -005 and -024 were tested in an assay as described in Ausiello etal., Tissue antigens 56, 538-547 (2000). Briefly, PBMCs were cultured at1×10⁶ cells/well in 48-well plates in the presence of 20 μg/ml ofantibodies and 10 ng/ml LPS (Sigma-Aldrich Chemie, Zwijndrecht, TheNetherlands) in 500 μl RPMI^(++.) After an O/N incubation at 37° C.,supernatant was harvested and stored at −20° C. The IL-6 concentrationwas assessed by ELISA (IL-6 ELISA kit, U-CyTech Biosciences, Utrecht,The Netherlands) according to the manufacturer's instructions. Data areshown mean concentration in pg/ml (±SEM) from 7 donors. The results showthat -003 and -005 does not induce release of significant IL-6 levels(FIG. 24B). Also -024 did not induce release of significant IL-6 levels(data not shown).

Example 20 Induction of Release of IFN-γ

-003, -005 and -024 were tested in an assay as described in Ausiello etal., Tissue antigens 56, 538-547 (2000). Briefly, PBMCs were cultured at1×10⁶ cells/well in 48-well plates in the presence of 20 μg/ml ofantibodies and 1 μg/ml OKT-3 (Sanquin, Amsterdam, The Netherlands) in500 μl RPMI^(++.) After an O/N incubation at 37° C., supernatant washarvested and stored at −20° C. The IFN-γ concentration was assessed byELISA (IFN-γ ELISA kit, U-CyTech Biosciences, Utrecht, The Netherlands)according to the manufacturer's instructions. Data are shown meanconcentration in pg/ml (±SEM) from 9 donors. The results show that -003and -005 does not induce release of detectable IFN-γ levels (FIG. 24C).Also -024 did not induce release of significant IFN-γ levels (data notshown).

Example 21 Affinity of Binding of -003 and -005 to Recombinant CD38

Binding of -003 and -005 to CD38 was tested using surface plasmonresonance. Briefly, purified antibodies were immobilized on a CM-5sensor chip (Biacore, Uppsala, Sweden) via anime coupling. HA-taggedCD38 (see Example 3) was flowed over, and the binding of antigen to mAbwas detected by a change in refractive index at the surface of the chipusing a Biacore 3000 (Biacore). The associated and rate constants for-003 (Table 7) and -005 (Table 8) are summarized below, mean of 3experiments ±SD, and show that both -003 and -005 have a high affinityfor CD38.

TABLE 7 Association and rate constants at 25° C. −003 k_(a) (1/Ms) 2.17× 10⁵ ± 2.65 × 10⁴ k_(d) (1/s)  1.9 × 10⁻⁴ ± 4.51 × 10⁻⁶ K_(A) (1/M)1.14 × 10⁹ ± 1.58 × 10⁸ K_(D) (M) 8.85 × 10⁻¹⁰ ± 1.2 × 10⁻¹⁰ 

TABLE 8 Association and rate constants at 25° C. −005 k_(a) (1/Ms) 8.88× 10⁴ ± 1.95 × 10⁴ k_(d) (1/s) 5.22 × 10⁻⁴ ± 1.16 × 10⁻⁵ K_(A) (1/M) 1.7 × 10⁸ ± 3.68 × 10⁷ K_(D) (M) 6.06 × 10⁻⁹ ± 1.21 × 10⁻⁹

Example 22 Epitope Mappinq

Epitope Mapping Using PEPSCAN Method

According to known procedures (Geysen et al. 1984. Use of peptidesynthesis to probe viral antigens for epitopes to a resolution of asingle amino acid. Proc Natl Acad Sci USA 81:3998; Slootstra et al.1996. Structural aspects of antibody-antigen interaction revealedthrough small random peptide libraries. Mol Divers 1:87; Puijk et al.2001. Segment synthesis. In PCT, The Netherlands, p.1.), overlapping20-mer linear and 15-mer looped peptides were synthesized covering 138amino acids at the C-terminus of human CD38. Furthermore, based on thesequence at the C-terminus single-looped peptides of different size weremade covering region KNIYRPDKFLQCVKNPEDSSCTSEI, regionCVHNLQPEKVQTLEAWVIHGG, and region CLESIISKRNIQFSAKNIYRC. In addition,extra sets were designed to reconstruct double-looped regions that werecomposed of SKRNIQFSCKNIYR and EKVQTLEAWVIHGG. Native cysteines werereplaced by alanines. Peptides were screened in an ELISA-assay usingcredit-card format mini-PEPSCAN cards.

Synthesis of Peptides

The peptides were synthesized using standard Fmoc-chemistry anddeprotected using TFA with scavengers. Subsequently, the deprotectedpeptides were reacted on the microarray with an 0.5 mM solution of2,6-bis(bromomethyl)pyridine or 2,4,6-tris(bromomethyl)mesitylene inammonium bicarbonate (20 mM, pH 7.9), supplemented with acetonitrile(1:1 [volume/volume]). The microarrays were gently shaken in thesolution for 30-60 min, while completely covered in the solution.Finally, the microarrays were washed extensively with excess ofMillipore H₂O and sonicated in disrupt-buffer containing 1% sodiumdodecylsulfate, 0.1% β-mercaptoethanol, in PBS (pH 7.2) at 70° C. for 30min, followed by sonication in millipore H₂O for another 45 min.

PEPSCAN ELISA-Assay

The 455-well credit card-format polyethylene cards, containing thecovalently linked peptides, were incubated with serum (e.g. diluted1:1000 in blocking solution which contains 5% horse serum[volume/volume] and 5% ovalbumin [weight/volume]) (4° C., overnight).After washing, the peptides were incubated with rabbit- anti-human Igperoxidase (dilution 1:1000, 25° C., 1 hour), and after washing theperoxidase substrate (2,2′-azino-di-3-ethylbenzthiazoline sulfonate and2 μl/ml 3% H₂O₂) was added. After one hour, the color development wasmeasured with a CCD-camera and an image processing system. The set upconsists of a CCD-camera with a 55 mm lens (Sony CCD Video CameraXC-77RR, Nikon micro-nikkor 55 mm f/2.8 lens), a camera adaptor (SonyCamera adaptor DC-77RR) and the Image Processing Software packageOptimas, version 6.5 (Media Cybernetics, Silver Spring, Md. 20910,U.S.A.; Optimas runs on a pentium II computer system).

Method for Epitope Representation

Individual amino acids were identified by dipeptide motifs whichrepresent the smallest unique units in the human CD38 amino acidsequence. All dipeptide motifs present in each of the 1164 peptidestested were awarded the ELISA value obtained for the respective wholepeptide. To rank the dipeptide motifs from strong to poor binding, arelative signal was calculated by dividing the ELISA value obtained foreach individual motif by the average ELISA value from all 1164 testedlinear and looped peptides, and these were sorted for decreasing values.In this manner, amino acid contributions to conformational epitopes wereconsidered. For each of the mAb tested, all dipeptide motifs scoringabove 2.5 (i.e. ELISA values of peptides containing these motifs were atleast 2.5 times the average ELISA value of those obtained with all 1164peptides) were selected. The data were de-convoluted into single aminoacid contributions represented on the linear CD38 sequence by a scoringsystem. By walking along the linear CD38 sequence and by using theunique dipeptide units as a reference point, one point was awarded eachtime a CD38 amino acid was present in this set of high scoring peptides.-003, 005 and -024 were all found to bind to the regions SKRNIQFSCKNIYRand EKVQTLEAWVIHGG of human CD38. -003 especially recognized the motifsRNIQF and WVIH, -005 especially recognized the motifs KRN and VQTL.

Example 23 Enzymatic Activity

The enzymatic activity of human CD38 was measured in an assayessentially as described in Graeff et al., J. Biol. Chem. 269,30260-30267 (1994). Briefly, substrate NGD⁺ (80 μM) was incubated withCD38 (0.6 μg/ml His-tagged extracellular domain of human CD38, seeExample 3 regarding purification of His-CD38) in a buffer containing 20mM Tris-HCl, pH 7.0. The production of cGDPR can be monitoredspectrophotometrically at the emission wavelength of 410 nm (excitationat 300 nm). In this example an excitation filter of 340±60 nm and anemission filter of 430±8 nm was used.

To test the effect of -003, -005 and -024 on the enzymatic activity ofCD38, recombinant His-CD38 protein was pre-incubated for 15 min at roomtemperature with various concentrations (30, 3, 0.3 and 0.03 μg/ml) ofthe different antibodies before adding the substrate NGD⁺. Theproduction of cyclic GDP-ribose (cGDPR) was recorded at different timepoints after addition of antibodies (3, 6, 9, 12, 30, 45, 60, 75 and 90min).

FIG. 25B shows that -005 has a pronounced inhibitory effect on theproduction of cGDPR. After 90 minutes, addition of 30 and 3 μg/ml -005resulted in a 32% and 34% reduced production of cGDPR (Table 9). Similarresults were observed in independent experiments using different batchesof -005.

No inhibitory effect on cGPDR production was observed after addition of-003 (FIG. 25B, Table 9), -024 (FIG. 25D, Table 9) or anti-KLH (FIG.25A, Table 9).

Based on these findings -005 is also expected to inhibit the synthesisof Cyclic ADP-ribose (cADPR) from NAD+. Inhibition of the synthesis ofcADPR can be determined according to the HPLC method described in Munshiet al., J. Biol. Chem. 275, 21566-21571 (2000).

TABLE 9 cGDPribose production in presence of CD38-specific antibodies oranti-KLH. Production (% of NGD control) 30 μg/ml 3 μg/ml 0.3 μg/ml 0.03μg/ml KLH 110 99 108 111 −003 99 100 107 107 −005 68 66 98 102 −024 99100 104 105

Example 24

Comparison of -003 and -005 with Morphosys Antibody 3079.

Antibodies -003 and -005 were functionally compared to Morphosysantibody 3079 (TH-3079). Methods for cloning and expression of Morphosysantibody TH-3079 are described in Example 16. Methods for CDC aredescribed in Example 6. Methods for ADCC are described in Example 5.FIG. 26A shows that -005 and -003 and TH-3079 induce CDC-mediated lysisof CD38-transfected CHO cells, with similar maximal lysis. When EC₅₀values are compared, -005 antibody is better than TH3079 in inducinglysis of CHO-CD38 cells, with 2-times lower EC₅₀ (see Table 10).

FIG. 26B shows that -005 is superior to TH-3079 in inducing CDC-mediatedlysis of Daudi-luciferase cells, with maximal lysis by -005 being 2-3times higher than by TH3079. When EC₅₀ values are compared, -005antibody is similar to TH-3079 in inducing lysis of Daudi-luciferasecells (see Table 10). -003 does not induce significant CDC-mediatedlysis of Daudi-luciferase cells.

FIG. 26C shows that in this experiment -005, -003 and TH-3079 mediatelysis of Daudi target cells via ADCC. No difference was found in (log)EC₅₀ and maximal lysis (Table 11, n=5).

TABLE 10 Maximal lysis and EC50 values of CD38-specific antibodies inCDC. CHO-CD38 cells (n = 2) Daudi-luc cells (n = 2) EC50 μg/ml % Max.lysis EC50 μg/ml % Max. lysis −005  0.15 ± 0.007 76.5 ± 3.54 0.39 ± 0.0070.5 ± 7.78 TH-3079  0.31 ± 0.021 81.5 ± 7.78 0.34 ± 0.26  25.5 ± 12.02−003  4.5 ± 0.933  62.0 ± 16.79 nc   12 ± 8.49

TABLE 11 Maximal lysis and EC₅₀ values of CD38 specific antibodies inADCC. Log STD log Maximal lysis STD EC50 EC50 (%) max. lysis −005 0.760.18 49.2 12.8 −003 1.17 0.23 64 14.2 TH3079 0.96 0.10 43.8 12.0

Example 25 Inhibition of Cellular Expressed CD38 Enzymatic Activity

The enzymatic activity of cellular expressed human CD38 was measured inan assay essentially as described in Graeff et al., J. Biol. Chem. 269,30260-30267 (1994). Briefly, substrate NGD (80 μM) was incubated with10⁵ CHO cells transfected with human CD38 (CHO-CD38 cells) in a buffercontaining 20 mM Tris-HCl, pH 7.0 supplemented with 30 μg/ml IgG1. Theproduction of cGDPR can be monitored spectrophotometrically at theemission wavelength of 410 nm (excitation at 300 nm). In this example anexcitation filter of 340±60 nm and an emission filter of 430±8 nm wasused.

To test the effect of -005 and -003 on the enzymatic activity ofcellular expressed CD38, CHO-CD38 cells were pre-incubated for 15′ atroom temp. with various concentrations (30, 3, 0.3 and 0.03 μg/ml) ofthe different antibodies before adding the substrate NGD. The productionof cGDPR was recorded at different time points after addition ofsubstrate NGD (3, 6, 9, 12, 30, 45, 60, 112 and 156 min).

After 156 minutes, addition of 30 and 3 μg/ml -005 resulted in a 21% and18% reduced production of cGDPR. No inhibitory effect on cGPDRproduction was observed after addition of -003 or IgG1 control antibody(Table 12).

TABLE 12 cGDPribose production in presence of CD38-specific antibodiesor IgG1 control. Production (% of NGD control) 30 μg/ml 3 μg/ml 0.3μg/ml 0.03 μg/ml IgG1 control 104 105 103 104 −003 107 106 107 105 −00579 82 100 104

Example 26

Binding of Antibody -005 to EBV Transformed Chimpanzee B Cells Afterharvesting and counting, EBV transformed chimpanzee B cells (receivedfrom Biomedical Primate Research Centre, Department Immunobiology,Rijswijk, The Netherlands) were resuspended (1×10⁶ cells/ml) in PBS-BSA(PBS supplemented with 0.1% BSA and 0.02% Na-azide). Then, cells wereput in 96-well V-bottom plates (100 μl/well) and washed twice inPBS-BSA. Thereafter, 50 μl FITC-labeled -005 antibody solution inPBS-BSA was added to the cells (4° C., 30 min). Cells were washed threetimes and specific binding of -005 to EBV transformed chimpanzee B cellswas detected by flow cytometry. FITC labeled HuMab-KLH (a humanmonoclonal antibody against KLH (keyhole limpet haemocyanin) generatedby Genmab B.V., Utrecht, The Netherlands by use of the immunizationprotocols described elsewhere herein) was used as a control. FIG. 27shows dose dependent binding of -005 to EBV transformed chimpanzee Bcells. No dose dependent binding to EBV transformed chimpanzee B cellswas observed with the control antibody HuMab-KLH.

Example 27

In Vitro Combination Therapy of Antibody -005 with Dexamethasone andBortezomib

Antibody -005 was tested for its capacity to induce cell death of themultiple myeloma cell line UM6 in vitro in a triple combination settingwith Dexamethasone (Dex) and Bortezomib (Bor; Velcade®). Outcome of thetriple treatment was compared to single drug treatments and double combotreatments.

3×10⁵ UM6 cells were incubated overnight at 37° C. with medium alone,with Dex (20 μM), with Bor (15 pM) or with the combination of Bor andDex. After 23 hours, -005 (10 μg/ml) was added, and 15 minutes afterthat, normal human serum was added and samples were incubated foranother 45 minutes at 37° C. Finally, 10 μl propidium iodide (PI;Sigma-Aldrich Chemie B.V.; 10 μg/ml) was added, and cell lysis wasdetected by flow cytometry using a FACS Calibur™ (Becton Dickinson) bymeasurement of the percentage of PI-positive cells.

As can be seen in FIG. 28, the triple treatment exceeded lysis observedwith any of the single or double combination treatments. This effect wasobserved in two independent experiments.

Example 28

Patients with a clinical diagnosis of multiple myeloma are treated witha combination of anti-CD38 antibody -005, melphalan and prednisone.

The compounds are administered to the patients according to thefollowing dosing schedule:

antibody -005: 8 mg/kg administered once weekly for 4 weeks (IV)

melphalan: 0.2 mg/kg per day IV for 4 days every 4-6 weeks

prednisone: 2 mg/kg PO for 4 days every 4-6 weeks

Response is determined by decrease in M-protein in serum, decrease innumber of plasma cells in bone marrow and decrease in Benze-Jonesprotein in urine and reduction of/absence of new osteolytic bonelesions.

Example 29

Patients with a clinical diagnosis of multiple myeloma are treated witha combination of anti-CD38 antibody -005, thalidomide and dexamethasone.

The compounds are administered to the patients according to thefollowing dosing schedule:

antibody -005: 8 mg/kg administered once weekly for 4 weeks (IV)

thalidomide: 200 mg/day (PO)

dexamethasone: 40 mg/day on day 1-4, 9-12 and 17-20 of each 28 day cycle(PO)

Response is determined by decrease in M-protein in serum, decrease innumber of plasma cells in bone marrow and decrease in Benze-Jonesprotein in urine and reduction of/absence of new osteolytic bonelesions.

Example 30

Patients with a clinical diagnosis of multiple myeloma are treated witha combination of anti-CD38 antibody -005, lenalidomide anddexamethasone.

The compounds are administered to the patients according to thefollowing dosing schedule:

antibody -005: 8 mg/kg administered once weekly for 4 weeks (IV)

lenalidomide: 25 mg/day (PO)

dexamethasone: 40 mg/day on day 1-4, 9-12 and 17-20 of each 28 day cycle(PO)

Response is determined by decrease in M-protein in serum, decrease innumber of plasma cells in bone marrow and decrease in Benze-Jonesprotein in urine and reduction of/absence of new osteolytic bonelesions.

Example 31

Patients with a clinical diagnosis of multiple myeloma are treated witha combination of anti-CD38 antibody -005, bortezomib and dexamethasone.

The compounds are administered to the patients according to thefollowing dosing schedule:

antibody -005: 8 mg/kg administered once weekly for 4 weeks (IV)

bortezomib: 1.3 mg/m2 on days 1, 4, 6 and 11, every 21 day cycle (IV)

dexamethasone: 40 mg/day on day 1-4, 9-12 and 17-20 of each 28 day cycle(PO)

Response is determined by decrease in M-protein in serum, decrease innumber of plasma cells in bone marrow and decrease in Benze-Jonesprotein in urine and reduction of/absence of new osteolytic bonelesions.

1-73. (canceled)
 74. A method for treating rheumatoid arthritis in asubject comprising administering to the subject an antibody which bindsto CD38 and comprises light and heavy chain variable region CDRs setforth in SEQ ID NOs: 13, 14, and 15 and SEQ ID NOs: 18, 19, and 20,respectively.
 75. A method for treating rheumatoid arthritis in asubject comprising administering to the subject an antibody which bindsto CD38 and comprises light and heavy chain variable region sequenceswhich are at least 90% identical to SEQ ID NOs: 12 and 17, respectively.76. The method of claim 74, wherein the antibody is internalized by CD38expressing cells.
 77. The method of claim 74, wherein said antibody doesnot induce release of significant IL-6 by human monocytes or peripheralblood mononuclear cells.
 78. The method of claim 74, wherein saidantibody does not induce release of detectable IFN-γ by human T cells orperipheral blood mononuclear cells.
 79. The method of claim 74, whereinthe antibody is a monoclonal antibody.
 80. The method of claim 74,wherein the antibody is a human monoclonal antibody.
 81. The method ofclaim 74, wherein the antibody is a full length IgG1, IgG2, IgG3, IgG4,IgD, IgA, IgE, or IgM antibody.
 82. The method of claim 74, wherein theantibody is an antibody fragment or a single-chain antibody.
 83. Themethod of claim 74, wherein the antibody is conjugated to a cytotoxicagent, a radioisotope, or a drug.
 84. The method of claim 74, furthercomprising administering at least one corticosteroid.
 85. The method ofclaim 74, further comprising administering an anti-inflammatory agent.86. The method of claim 74, further comprising administering animmunosuppressive agent.
 87. The method of claim 74, further comprisingadministering an immunomodulatory agent.
 88. A method for treatingmultiple myeloma in a subject comprising administering to the subject anantibody which binds to CD38 and comprises light and heavy chainvariable region CDRs set forth in SEQ ID NOs: 13, 14, and 15 and SEQ IDNOs: 18, 19, and 20, respectively.